WO2014201308A1

WO2014201308A1 - Endothelial-targeted adenoviral vectors, methods and uses therefor

Info

Publication number: WO2014201308A1
Application number: PCT/US2014/042204
Authority: WO
Inventors: Jeffrey ARBEIT; David Curiel
Original assignee: Washington University
Priority date: 2013-06-12
Filing date: 2014-06-12
Publication date: 2014-12-18
Also published as: US20170159072A9; US20160145643A1

Abstract

Disclosed are adenovirus vectors comprising a ROBO4 enhancer/promoter operatively linked to a transgene. Also disclosed are adenovirus vectors comprising a chimeric AD5-T4 phage fibritin shaft, a trimerization domain displaying a myeloid cell-binding peptide (MBP), and a ROBO4 enhancer/promoter operatively linked to a transgene. Also disclosed are methods of expressing a transgene in an endothelial cell in vivo, comprising administering to a mammal an adenovirus comprising a ROBO4 enhancer/promoter operatively linked to a transgene. Also disclosed are uses of the adenoviral vectors, including mobilization of granulocytes, monocytes and lymphocytes from bone marrow, mobilization of cancer cells in vivo, selective targeting of endothelial cells, and cancer treatment methods.

Description

Bndothelial-utrgeted Adenoviral Vectors, Methods and Uses Therefor

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority from U.S. Provisional. Application Serial No.

61/834,385 filed June 12, 2013, which is incorporated herein by reference in its entirety,

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This work received support from Nlii EtfilCA 159959, and CA.154697. The government may have certain rights in the invention.

Introduction

in stem cell biology, stem cells possess the properties of self-renewal, proliferati ve quiescence, and organ/tumor multi-lineage repopuiaiion (Barker ei al 2010). Stem cells can require a host cellular niche to maintain their functions (Voog and Jones 2010). Stent ceils in most organs and tissues persist for the organism lifetime (Voog and Jones 2010). Persistence can be. due to markedly prolonged cell cycle times inferred by prolonged retentio of the nucleotide analogs tritiaied thymidine or hro odeexyuridine (BrdU), or assayed by chromatin bound histone 2B fluorophore fusion proteins (Foudi t al 2009). Tissue stem cell biology has been conceptually modeled based on the hierarchical organization of stem and progenitor ceils in the hematopoietic; system (Essers and Trarapp 2010), A hematopoietic cellular hierarchy has been identified and stem cells isolated by fluorescence activated cell sorting of cell surface markers combined with functional cell culture and intact animal repopuiation and colony forming assays (Rieger and Schroeder 2012: May le ei al 2013).

Existence of prostate stem ceils, in particular in the roden t, was strongly suggested by studies demonstrating 30 cycles of prostate regeneration following castration and exogenous androgen provision (English ei l 1987), A combination of flow cytometric markers, serial cell culture, and tissue recombination with kidney subcapsular grafting enabled construction of murine and human prostate stem/differentiated cell hierarchies (Goldstein and Witte 2012; Goo ei al 2012). Prostate stem like cells form spheroids "prostaspheres" when grown, in anchorage-independent ceil culture (Lofcaes et al 2010; Rhi.m 201 ). Prostasplieres self- renew during prolonged serial passage, and repopuiate tubules and duets, forming prostate organoids when re-implanted into mice (Azum& ei al 2005; Goo ei al 2012). Stem cells have been, identified in classical PC A ceil lines including PC3, DO 144, parental LNCaP, and derivative LNCaP~2B cells (Miki ei al 2007).

Approaches targeting metastatic neovasculature are needed. An option can. be tumor vascular endothelial cell (EC) adenoviral (Ad) vector targeting. Although EC transductional and transcriptional targeting has been accomplished, vector administration, approaches of limited clinical utility, lack of tumor-wide EC expression quantification, and a failure to address avid liver sequestration, has challenged, prior research. Previous vascular targeted drugs and biologies aim to destroy/inhibit the formation of new vasculature in an attempt to inhibit either tumor growth or subdue inflammation.

The tumor neovascularization field remains challenged, by the multiple evasion mechanisms induced in malignancies during antiangiogenic therapies (Bergers G et al 2008).. The discovery of vascular endothelial growth factor (VEGF) (Ferrara N 2004) and VEGF delineation as one of the predominant tumor produced angiogenic, factors spawned research into drugs and biologies targeting tumor production, stromal availability, and VEGF receptor signal transduction i^'Cook KM et al 2010). Although some patients experience tumor size reductions from available methods, tumor growth eventually resumes. De novo or acquired tumor antiangiogenic therapy resistance can be due to several factors. One evasion mechanism is cancer ceil, production of untargeted angiogenic factors (Bergers G et al. 2008). Another mechanism is tumor chemo and cy tokine endocrine secretion that mobilizes and recruits proangiogenic bone marrow myeloid and immune cells (Ferrara N 2010). T us.nor- aetivated stromal fibroblasts can produce untargeted. angiogenic factors. (Crawford. Y ef al 2009). Tumors can also shift their growth patterns and invade into tissues by host blood vessel cooption (Leenders WP et al 2004).

There Iras been a research interest in targeting tumor neovascularization (Goldman ei al 1998; Triosszi and Borden. 201 1). A series of papers have indirectly targeted neovesseis by vectors engineered for tumor cellular expression of soluble angiogenesis growth factor decoys (Mahasreshii et al 2001 ), Another approach focused o neovessel transduction using capsid display of peptides cognate for receptors upreguiated on tumor microvessels

(Bachtarzi et al 2011). Another strategy used enhancer/promoters differentially activated in tumor ECs by the tumor m croenvimnment (Jaggar ?/ al \ 997; Takayama et al 2007; Dong and Nor 2009). Yet the goal of these past studies was to eradicate tumor neovesseis. There has also been research interest in tissue resident stem ceils beyond those known to mpopuiate rapid turnover organs such as the gut and skin (Barker et al 2010). Fast research has also u tilized a "typical" cytolytic or apoptotic vector approach of conditionally replicating Ad CCRAd) vectors.

Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Tumor vasculature can be a conduit for nutrient and oxygen influx and metabolic efflux, however emerging studies demonstrate that the microvascalature and the vascular endothelial cell (EC), can be components for estabhshment and maintenance of niches for host organ stem cells (Ding L ei al. 2012), Tumor stem/initiating cells have been identified in these perivascular niches ( hu TS e( al 20! I). The perivascular niche can be maintained by short range, "angiocrine", EC growth factor secretion and contact between tumor cells and host microvessels (Butler JM et al. 2 10),

The tumor gene therapy field is challenged by several issues; target cell vector transduction, hepatic toxicity due to viral gene expression, and innate arid adaptive host vector immune response ( hare R et « , 201 1 ; Duffy MR ef al. 2012), Previous studies have failed to investigate vector vascular expression in an extensive panel of host organs, and elucidate global determination of reporter expression distribution throughout the tumor neevascuiature.

Gene therapy approaches to the vascular endothelium have exploited several approaches. Vector-host cell transduction was manipulated to produce tumor EC targeting (Reynolds PN ei al. 2000; Baker All ei al. 2005), Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgetie pavioad capacity and lack of somatic mutation risk, Adenovirai and adeno-assoeiated vectors have been engineered for eapsi. display of peptides identified, on tumor-activated

endothelium,, or hispecific antibodies cognate for mtegrins, se!ectins, or vessel luminal cell surface receptors (Preuss MA ei al. 2008; Bachtarei H el al 201 I ; Nette!beck DM el al 2001 ). Vector pseudotyping using ftberknobs from serotypes other than adenovirus type 5, other animal host species, or fiber replacements, either from other viruses or viru -synthetic chimeric fibers, also achieved EC tropism (Preuss MA e-t l. 2008; Shinostaki ei al 2006). However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration.

Some studies have focused on the dual goals of liver sequestration inhibition, and hCAR de-targeting concomitant with tumor EC transductions! targeting ( Bachtarzi H ei al 201 1). ConditionaHy replicaiive adenoviral vectors were used based on tumor angiogenic .factor induced EC proliferation (Peled M el al. 2009; Takayama K. et al. 2007), Other efforts centered on transcriptional targeting using DNA enhancer/promote.- elements induced in tumor-activated EC's either due to growth factor stimulation or tumor microenvironmental alterations such as hypoxia (Dong Z et al. 2009; Greenberger S et al. 2004; Savon tans MJ ef al. 200.2). A receni study revealed that human "'androgen independent" PCA CSCs can he segregated based on a PSA "low" reporter expression (Qin et al 2012), Another recent study discovered that PCA stem like cells directly home to the bone marrow (BM) hematopoietic stem cell (HSC) niche, PCA stem cells both physically and biochemically mobilized PfSCs out of the niche into the more differentiated hematopoieti progenitor cell (HPC) poo!

(Shiozawa et al 2011). This PCA. stem ceil HSC eviction .function was controlled by ceil surface CXCR4. Abrogatio of PCA stem cell CXCR4-bone marrow niche SDF ! adherence by the CXCR4 blocker, ΑΜΌ3100 mobilized PCA CSCs into the circulation. Beyond SDF 1 - CXCR4, other iigand receptoi^" signaling modules such as W t- Frizzled receptor, delta/jagged family Notch ligands/receptors, and sonic hedgehog-patched have all been implicated in. PCA metastatic growth (Leong and Gao 2008; Takebe el al. 201 1 ). l.igand decoys have been generated for many of these receptors (Funahashi el al 2008; Lavergite el al 201.1 }. Despite the extensive research on PCA CSC isolation and function, u!ti -directional interactions between the melange of bone niche cellular components regulating CSC maintenance, and metastatic PCA growth have not been investigated in depth.

PCA cells can reach the bone via several routes, in BM, PCA cells can adhere to and traverse sinusoidal ECs (G^'linsky 2006). PCA.-EC adherence can to be regulated by a combination of integrin αν β3 and CXC.R4 chemokine receptor engagement and signaling. PCA cells express CXCR.4 and bone perivascular stromal, ceils, sinusoidal ECs, osteoblasts, and mesenchymal cells express the CXC.R4 llganci, stromal derived factor-! , SDF- 1/C.XCLI2. Bone colonizing PCA ceils can also engage a gene expression program termed, "osteogenic mimicry" (Chung et al. 2009). PCA. cells can upregulaie molecules activatin both osteoclasts and osteoblasts. Receptor activator of NFkB Hgand ( AN L) can engage its RAN receptor on osteoclasts to stimulate bone resorption. PCA parathyroid hormone production can similarly stimulates osteoclasts (Kostenuik. et al. 2009). Osteoclastogenesis enhanced bone resorption can release bone matrix bound growth factors such as. TGFji thai activate both PCA growth and. expansion and osteoblasts to produce bone matrix, leading to increased though abnormal woven bone formation (Ibrahim et al 2010), Molecules

stimulating angiogeuesis such as VEGf and basic FGF can be released by osteoclasts from the bone matrix, and from metastatic PCA cells (Morrissey et af 2008}.. Collectively, the growth factor/eheraokme rich metastatic bone mieroenvironraent can enhance proliferation and npregu.iate survival pathways that can facilitate PCA chemotherapeutic resistance (Sotmik ei ai 201 1 >. CSC mobilization, has een achieved using small molecule receptor inhibitors, but the effect is global rather than niche targeted. Drugs such as AMD3100 are well tolerated but. present the specter of indiscrimmant ESC mobilization complicating tandem cytotoxic chemotherapy administration. Enhanced bone metastatic tumor growth dae io AMD310Q- mediated osieoclasiogenesis induction is another example of global off-target effects of systemic administration of stem cell ligand blocking factors (Hirhe et al 2007).

PCA CSCs can compete with host HSPCs for BM niches (Shiozawa ei «/. 201 1).. Recent work used lineage-marked mice to elucidate the specific cell types controlling host HSPC -maintenance (Nagasawa ei ah 201 1 ; Ding and Morrison 20 i 3; Oreenbaum ei al 2013). Lineage tracing has yet to be extensively used to study PCA. CSC niche interactions. The cellular niche organization and anatomical relationships of the BM have been recently elucidated.. There is a close juxtaposition and/or encirclement of host sinusoidal capillaries by niche components (Nagasawa ei «/. 201. 1 ).

Past research using antibodies and small, molecule drags has focused, on. ablating or inhibiting the proces of tumor neovascularization to starve a tumor of nutrients and oxygen. However, tumors possess multiple redundant pathways evading neovascular ablation. Hence, these strategies have in general failed to achieve survival benefits for cancer patients. Nor does the vascular ablation approach benefit patients with benign but equally morbid or lethal diseases such as autoimmune inflammatory diseases, bone marrow failure, Alzheimer's, amyotrophic lateral sclerosis, or multiple sclerosis.

US Patent Application 2006/0099.1 3, "Antibodies Binding to ^'Human Magic Roundabout (MR), Polypeptides and Uses Thereof for inhibition Angiogensis," {Bicknel! ei al.) describes an antibody that binds to Magic Roundabout and an expression system in a host cell using an adenovirus, with a promoter. The expression system encodes a polynucleotide in a suitable host cell to produce the antibody or compound of the invention to inhibit angiogenesis and all diseases associated with angiogenesis using expression of a decoy fragment of the Magic ^'Roundabout (ROB0 ) protein. However, this reference is silent about the R.OB04 promoter.

US Patent Application 2010/0222401, "Compositions and Methods for Treating Pathologic Angiogenesis and Vascular Permeability " (Li et al) describes methods for producing and screening compounds and compositions capable of modulating the described signaling pathway, inhibiting vascular permeability, and inhibiting pathologic angiogenesis. The signaling pathway described in the application is Robo4 signaling and its ability to inhibit protrusive events involved in cell migration, stabilize endothelial cell-cell junctions, and block pathological angiogenesis_* This application discloses that expressing .Robo4 using an adenoviral vector and Robo4's expression is endothelial-speciffc. This application does not teach using an adenoviral vector to target expression to endothelial cells by use of the Roho4 promoter/enhancer fragment.

US Patent 8,3943 1₁ "Antibodies, polypeptides and uses thereof" (Bicknell ei al.) discloses a method of inhibiting angiogenesis in an individual in need thereof by

administering an antibody that binds Magic Roundabout (MR) or a fragment thereof thai inhibits its endothelial ce!i migration and/or proliferation.

"A three-kilobase fragment of the human Roho4 promoter directs cell type-specific expression in endothelium,'^* (Ofcada et al. Cir. Res. 100: 1712- 1722, 200?) describes the use of the Robo4 promoter for targeting gene expression from vectors to the endothelial cells.

"Derivation of a myeloid cell-binding adenovirus for gene therapy of inflammation," Alberts, M.O., et a)., PLoS one7:e37812₅ 2012} discloses an adenovirus comprising BP. and binding of viruses to primary myeloid cell types. Binding is illustrated for peripheral blood, spleen and lung myeloid cells. However, viral transduction or expression in endothelial cells is .not disclosed.

A goal of past vascular- targeted therapies was intratumoral ablation in order to ""starve" the tumor of nutrients^' and oxygen. However, vessel ablative therapies can render the tumor microenvironment hypoxic redox stressed. This altered microenvironmeai can produce untargeted angiogenic factors either via malignant cell autocrine production, or from host bone marrow (BM) derived cells recruited by endocrine tumoral production. The efficac of intratumoral vessel "normalization" to increase perfusion and consequently drug and oxygen, delivery tor enhanced radiosensitivity has been questioned by recent studies.

The targeting efficacies and the therapeutic utility of these approaches were affected by different factors. Some studies were solely performed in cultured EC^'s ( ettelbeck DM ei al. 2001 ; Yang WY ei til 2006). Bridging studies tested in vitro transduced ECs in mixed tumor-EC injections (Mavria G ei al 2000). Other approaches used direct injection of vascular-targeted vectors into tumors (Song W ei al, 2008). These experimental strategies failed to address the crucial challenge of tumor vessel delivery following systemic administration that is the preclinical translations! lynchpm. Prior work engaging systemic vector deliver)-' predominantly used enzymatic luciferase assays of whole tissue that were not linearly quantitative (Takaya a K. t aL 2007). Studies documenting co-locaiization frequently presented "coned down." high magnification views of single vessels but failed to evaluate tumor-wide vascular distribution (Baehtara H al 2011 ; Varda-Bloom N et al 200 l ; Hais«ia HJ 2010).

The concept of enzymatic conversion of an inactive prodrug to an active derivative has been employed in cancer therapeutics. One approach has been to capitaliz on host cellular, or cancer cell overexpression of endogenous prodrug convening enzymes, such as thymidy!ate kinase for eapeeitabine conversion to DNA RNA nucleotide 5-FU or carboxyl esterase mnotecan conversion to the active topoisomerase inhibitor S 38 (Rivory et al. 96; Hatfield et al 201 i ; Shindoh et al 201 1 ). Another approach has been to transduce tumor cells directly by local injection of Ad vectors encoding prodrugconverting enzymes such as thymidine kinase or eytosine deaminase (CD) (Kaiiherov et al. 2006; Fuchita ei. al. 2009).

Subsequent studies also detected ROB04 activation in lymphatic endothelium and in hematopoietic stem cells (Smith-Berdan S et 2011; Zhang X et 2012). RO.B04 function has been controversial ranging front angiogenesis in zebrafish (Bedell VM ei al 2005). or negative regulation, in the mammary gland (Marlow R et al 2010), to vascular integrity and stabilization (Jones CA et al 2008), migration inhibition (Park KW et al 2003) versus stimulation (Sheldon !i ei al. 2009), and repulsion (Koch AW et al 20.1 i). At the molecular level, ROB04 was shown to bind paxil!in leading to inhibition of Rac activation and !ameUipodial formation via ΟΪΤ.1 -GAP Arf6 GT.Pase Inactivaiion (Jones CA et al 2009). Most of the R.OB04 functions were delineated using Slit proteins as presumptive ligands {Jones CA ei al 2008), however more recent work definitively demonstrated (lie U C5B receptor as the ROB04 binding partner (Koch AW e al 201 1).

Prior research has undergone challenges in discerning the differential upregulation of endogenous ROB04 expression in tumor activated versus quiescent endothelium because most localization studies have used enzyme reporter genes (Okada Y ei al 2007; Jones CA ei al 2008), though ROB04 has been implicated as a marker of activated endotheliu

(Huroiniecki L ei a 2002; Seth P ei al. 2005).

Summary

The present inventors have developed methods and compositions that make use of the intact vasculature and the endothelial cells (ECs) contained therein as vehicles for delivery of therapeutic agents in benign and malignant disease. In various embodiments, adenoviral vectors are targeted to vascular endothelial cells, in some configurations, the endothelial cell- targeted adenoviral vectors can provide angiocrine functions and thus can be used to treat malignant and benign diseases. In various embodiments, transgene-carrying adenoviral vectors of the. present teachings include the following: 1 ) adenoviral vectors which selectively

? enter (transduce) and/or are exclusively expressed in vascular ECs; 2) adenoviral vectors comprising transgenes which encode prodrug converting enzymes which produce active cytotoxic chemotherapy drugs following inactive prodrug administration 3) adenoviral vectors comprising transgenes that convert prodrugs or elaborate conversion product molecules that are secreted by ECs into the tissue mieroenvironments, 4) adenoviral vectors comprising transgenes that ate expressed in ECs and activate EC surface molecules to affect cellular function in an adjacent mieroemdronment, 5) adenoviral vectors comprising

transgenes that inhibit inflammation by sequestration of chemo- ox cytokines, or encode molecules stimulating disaggregation of plaque formation in Alzheimer's or other benign diseases.

in various embodiments, the present Cachings make use of the fact that the

vasculature provides widespread, access to diseased tissue. In addition, the vascular endothelial cells are in close approximation of target cells within diseased tissue allowing increased and more speci fic targeted dosing of therapeutic agents. Furthermore, the vascular endothelium is the first cell type/organ encountered by adenoviral vectors. Thus, systemic intravenous or intraarterial vector injection can target vascular endothelium prior to uptake in nonvascular cells in organs and tissues. In some embodiments, endothelial targeted adenoviral vectors can he engineered for cargo gene expression that can be restricted to di ease tissue mieroenvironments. The microenvironment can include different cell types in additton to the diseased celis. .Ancillary cell types can include fibroblasts, inflammatory cells- myeloid cells, macrophages and lymphocytes, and. fibroblasts. Collectively the crosstalk between diseased cells and the ancillary cellular collection can chang the tissue

niicroenvironnient. Such changes can include low oxygen, low pH- high acidity, altered redox potential and intracellular stress. There can also be DMA regulatory regions- enhancer/promoters that, are solely activated by one or more diseased tissue

microenvironmental alterations, in various configurations, enhancer/promoters can be engineered into adenoviral vectors to increase transgene expression in diseased compared to normal tissue specificity,

in various embodiments, endofhelial-targeied adenoviral vectors of the present teachings can be applied to a variety of diseases, including, without limitation, the following:

Cancer, such as solid organ primary si te (site of origin) cancer, in particular brain cancer; solid organ metastatic cancer, including but not limited to bone, lung, liver, and lymph nodes, occult cancer metastatic imaging, hematopoietic cancers, including multiple myeloma, leukemia, lymphoma. Benign diseases., such as inflammatory diseases including but not limited to rheumatoid arthritis, atherosclerosis, psoriasis, Crohn's disease, ulcerative colitis. Type 1 {juvenile onset) diabetes, inflammatory and degenerative central nervous system diseases including but .not limited to: Alzheimer's disease, multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis; osteoporosis via endothelial angiocrine osteoclast inhibition alone or combined with concomitant angioerme osteoblast stimulation, vascular

msuffieiency iscnemic disease including but not limited to: coronary artery disease, lower limb arteriosclerotic vascular iiisotTicseiicy (peripheral vascular disease), ischemic stroke, CNS diseases including but not limited to cerebral vasospasm following subarachnoid hemorrhage.

in various embodiments, the present teachings include an adenovirus vector comprising a ROB04 enhancer/promoter operativeiy linked to a transgene. in various configurations, the transgene can encode a prodrug converting enzyme. In various configurations, the prodrug converting enzyme can be a ey^'tosine deaminase. In various configurations, the transgene can encode a decoy receptor, such as, without limitation, a decoy receptor that binds at least one angiocrine factor. In various aspects, the transgene can encode a truncated CXCR4 receptor. In some configurations, a ROB04 enhancer/promoter of the present teachings can comprise a tissue-specific expression, control element. In some configurations, a ROB04 enhancer/promoter of the present teachings can comprise a let response element. In some configurations, a ROB04 enhancer/promoter of the present teachings can comprise a hypoxia-response element. In some configurations, a ROB04 enhancer/promoter of the present teachings can. comprise a GABP-biading element

in various embodiments, the present teachings include an adenovirus vector comprising a chimeric AD5-T4 phage ilbritsn shaft, a trimeri ation domain displaying a heptapepide, "myeloid cell-binding peptide" (MBP), and a OB04 enhancer/promoter operativeiy linked to a transgene. Ad.MBP includes MBP displayed at the tip of a "de- knobbed" chimeric fiber (Muro, S„ et al 2004; Alberti, MX⁾., et al 2032). This vector was shown to bind specifically to myeloid cells ex vivo but predominantly transduced long vascular endothelium following systemic administration. (Alberti, M.O., et al. 2013). In various configurations, me transgene can encode, without limitation, a reporter, such as a green fluorescent protein, or a prodrug converting enzyme, such, as, without limitation, a cytosine deaminase, in various configurations, the transgene can encode a decoy receptor, such as, without limitation, a decoy receptor that binds at least one angiocrine factor. In various configurations, the transgene can encode a truncated CXCR4 receptor. In various embodiments,, an ..Ad.MBP of the present teachings can provide widespread EC transduction in organs such as long, ^'heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Accordingly, in some embodiments, the present teachings provide molecular access to hitherto inaccessible organs including brain, small and large b wel mucosa, kidney glomeruli, medulla, and papilla, skeletal muscle, and cardiac subeodocatclium and

myocardium. Thus, in various embodiments, a vector of the present ieachings can be used for targeting many prominent and vexing human diseases.

i n various configurations, Ad.MBP can retain hepatocyte tropis n albeit at a reduced frequency compared with standard AdS. in various configurations, Ad.MBP can bind specifically to myeloid cells ex vivo, in various configurations, multi-organ Ad.MBP expression is not dependent on circulating monocytes or macrophages. In various configurations, Ad.MBP dose de-escalation can maintain, full lung targeting capacity but drastically reduced transgene expression in other organs, in variou configurations, swapping the EC-specific R0BO4 promoter for the CM V promoter/enhancer can abrogate hepatocy e expression and can also reduce gene expression in other organs.

I.n various embodiments, the present teachings incl ude methods of expressing a transgene in an endothelial ceil (EC) in vivo. In various configurations, the methods can comprise administering to a mammal an adenovirus comprising a RO 04 enhancer/promoter operativeSy linked to a transgene. In various aspects, the transgene can encode a prodrug converting enzyme, such as, without limitation, a cyiosine deaminase. In various aspects, the transgene can encode a decoy receptor, uch as, without limitation, a decoy receptor that binds at least one angiocrine factor. In various aspects, the transgene can encode a truncated CXCR4 receptor.

In various embodiments, the present teachings include methods of mobilizing at least one of granulocytes, monocytes and lymphocytes from bone marrow. In various

con figurations, these methods can include administering to a mammal an adenovirus comprising a ROB04 enhancer/promoter operationally linked to a transgene encoding a truncated XCR4 receptor,

in various embodiments, the present teachings include methods of mobilizing cancer cells in vivo. In various configurations, these methods can include administering to a mammal an adenovirus comprising a ROB04 enhancer/promoter operationally linked to a transgene encoding a truncated CXCR4 receptor. In various aspects, the cancer cells can be comprised by bone marrow (BM). In various embodiments,, the present teachings include methods of selectively targeting endothelial cells. 3n various configurations, these methods can comprise administering to a mammal an adenovirus, wherein the adenovirus comprises a chimeric AD5-T4 phage fibritin shaft and a trimerixation domain displaying a myeloid cell-binding peptide (MBP), and an exogenous promoter operative!}' linked to a transgene. in various configurations, the exogenous promoter can be or can comprise or consist of a ROB04 enhancer/promoter. In various configurations, the exogenous promoter can be or can comprise or consist of a Tel-respon ive element in various configurations, the exogenous promoter can be or can comprise or consist of a aypoxia-responsive element, in various configurations, the endothelial cells (ECs) can be selected from the group consisting of brain ECs, kidney ECs and muscle ECs. In various configurations, the transgene can encode a. truncated CXCR4 receptor.

in various embodiments, the present teachings include methods of treating a cancer, in various configurations, these methods can comprise administering to a mammal an adenovirus comprising a chimeric A.D5-T4 phage fibritin shaft and trimerization domain displaying a myeloid cell-binding pteptide (MBP) and a nucleic acid sequence encoding a truncated CXC 4 receptor, and administering.a chemotherapeutic agent, in various configurations, the administration of a chemotherapeutic agent can comprise or consist of administering a therapeutically effective, amount of the chemotherapeutic agent

in various embodiments, the present teachings include use of an adenovirus vector comprising a ROB04 enhaneer promotar operattvely linked to a transgene for the treatment of a disease such as, without limitation, a cancer, such as solid organ primary site ( site of origin) cancer, in particular brain cancer; solid organ metastatic cancer: including but not limited to bone, lung, liver, and lymph nodes; occult cancer metastatic imaging;

hematopoietic cancers, including .multiple myeloma, leukemia, or lymphoma. In various embodiments, the present teachings include use of an adenovirus vector comprising a ROB04 enhancer/promoter operative!}' linked to a transgene for the treatment of a disease such as, without, limitation, a benign disease, such as, without limitation, an inflammatory disease such as rheumatoid arthritis, atherosclerosis, psoriasis, Crohn's disease, ulcerative colitis. Type 1 (ju venile onset) or diabetes. In various embodiments, the present teachings include use of an adenovirus vector comprising a OB04 enhancer/promoter operatt vely linked to a transgene for the treatment of a disease such as, without limitation, an inflammator and degenerative central nervous system disease such as Alzheimer's disease, multiple sclerosis, Parkinson's disease or amyotrophic lateral sclerosis. In various

Π embodiments, the present teachings include use of an. adenovirus vector comprising a

OB04 enhancer/promoter operativeiy linked to a transgene for the treatment of a disease such as, without limitation, osteoporosis via endothelial angioerine osteoclast inhibition alone or combined with concomitant angioerine osteoblast stimulation. In various embedments, th e present teachings include use of an adenovirus vector comprising a ROB04

enhancer promoter operativeiy Jinked to a transgene for the treatment of a disease such as, without limitation, a vascular msufficiency/tschemic- disease such as coronary artery disease, lower limb arteriosclerotic vascular insufficiency (peripheral vasculai disease), or ischemic stroke, in various embodiments, the present teachings include use of as adenovirus vector comprising a ROB04 enhancer/promoter operativeiy linked to a transgene for the treatment of a disease such as, without limitation, a CNS disease such as cerebral vasospasm, following subarachnoid hemorrhage.

in various embodiments, the present teachings include methods of treating a disease or disorder that^" activates angiogenesis in. villous endothelium. In various configurations, these methods can comprise administering to a mammal an adenovirus vector comprising a ROB04 enhancer/promoter operativeiy linked to a transgene. in some configurations, a disease or disorder of these embodiments can be selected, from the group consisting of inflammatory bowel disease regional enteritis, inflammatory bowel disease of the colon, infection with toxin producing bacteria, and colon cancer- recursor legions of multiple polyposis. In some aspects, a transgene of these embodiments can encode a secreted antiinflammatory cytokine decoy. In some aspects, a decoy can be selected from the group consisting of soluble TNF-alpha receptor, single chain aori-!LI , single chain anti-ILl? antibody, a bacterial anti-toxin, and an RNAi molecule targeting gene product induced by the activation of the WNT pathway in multiple polyposis, in some configurations, the toxin- producing bacteria can be selected from the group consisting of Clostridium difficile,

Chstridmm boiu!immh and Shigella,

in some embodiments, the present teachings disclose-methods of^" treating an inflammatory CNS disease in a mammal, in various configurations, these methods can comprise administering to the mammal an Ad.MBP.CMV vector encoding a cytokine decoy. In various configurations, the- inflammatory disease can be selected from the group consisting of amyotrophic lateral sclerosis and multiple sclerosis.

In some embodiments, the present teachings disclose methods of treating a degenerative disease in a mammal In various configurations, these methods can comprise administering to the mammal an Ad.MBP.CMV vector encoding a cytokine decoy. In various aspects, the degenerative disease can be selected from the group consisting of Alzheimer's disease and Parkinson's disease.

In some embodiments, the present teachings disclose methods of stimulating appetite in a mammal In various configurations, these methods can comprise administerin to the mammal an A&MBP.CMV vector encoding a secreted molecule thai .affects the

hypothalamic appetite nuclei,

in some embodiments, the present teachings disclose methods of inducing satiety in a mammal, in various configurations, these methods can comprise administering to the mammal an Ad.MBP.C V vector encoding a secreted molecule that affects the

hypotli.aia.mic appetite nuclei.

In some embodiments, the present teachings disclose methods of treating

myelodysplasia syndrome in a mammal In various configurations, these methods can comprise administering to the .mammal an Ad.RGD.H5/H3.RQB04 vector, wherein the Ad.RGD.M5/M3.R08CM vector produces at least one anti-inflammatory molecule.

In some embodiments, the present teachings disclose methods of treating a genetic disease selected from the group consisting of hemophilia and sickle ceil anemia io a raattimal in various configurations, these methods can comprise administering to the mammal an Ad.RGD.H5/H3 JROB04 vector, wherein the Ad.RGD.HS/H3.ROB04 vector produces at least one anti-in ammatory molecule.

In some embodiments, the present teachings disclose methods of treating a cancer in a mammal, in various configurations, these methods can comprise admi istering to the mammal an Ad.RGD,H5/H3..ROB04 vector, wherein the Ad,RGD.H5/f-B.ROB04 vector produces at least one molecule selected front the group consisting of a .molecule that mobilizes metastatic cancer or leukemic stem cells and a. molecule producing a

chemoihetapeutic prodrug converting enzyme.

In various embodiments, the present teachings include an adenovirus vector comprising a ROB04 enhancer/promoter operativeiv linked to a transgene for use in the treatment of a disease such as, without limitation, a cancer, such as solid organ primary site (site of origin) cancer, in particular brain cancer; solid organ metastatic cancer: including but not limited to bone, lung, liver, and lymph nodes; occult cancer metastatic imaging;

hematopoietic cancers, including multiple myeloma, leukemia, or lymphoma. In various embodiments, the present teachings include an adenovirus vector comprising a ROB04 enhancer/promoter operativeiv linked to a transgene for use io the treatment of a disease such, as, without limitation, a benign disease, such as, without limitation, an inflammatory disease such as rheumatoid arthritis., atherosclerosis, psoriasis, Crohn's disease, ulcerative colitis. Type 1 (iuveniSe onset_.) or diabetes. In various embodiments, the present teachings include an adenovirus vector comprising a ROB04 enhancer/promoter operatively linked to a transgene for use in the treatment of a disease such as, without limitation, an inflammatory and degenerative central nervous system disease such as Alzheimer's disease, multiple sclerosis, Parkinson's disease or amyotrophic lateral sclerosis. In various embodiments, the present teachings include an adenovirus vector comprising a. R08O4 enhancer/promoter operatively linked to a transgene for use in the treatment of a disease such as, without limitation, osteoporosis via endothelial angiocrine osteoclast inhibition alone or combined wit concomitant angioerine osteoblast stimulation. In various embodiments, the present teachings include an adenovirus vector comprising a ROB04 enhancer/promoter operatively linked to a transgene for use in the treatment of a disease such as, w i thout limitation, a vascular insufllcteney/ischemic disease such as coronary artery disease, lower limb arteriosclerotic vascular insufficiency (peripheral vascular disease), or ischemic stroke. In various embodiments, the present teachings include an adenovirus vector comprising a ROB04 enhancer/promoter operatively linked to a transgene for use in the treatment of a disease such as, without limitation, a C S disease such as cerebral vasospasm following subarachnoid hemorrhage.

in various embodiments, the present teachings include use of an adenovirus v ec tor comprising a OB04 enhancer/promoter operatively linked to a transgene for the manufacture of a medicament to treat a disease such as, without limitation, a cancer, such as solid organ primary site (site of origin) cancer, in particular brain cancer, a solid organ metastatic cancer including but not limited to bone, lung, li er, and lymph nodes, occult cancer metastatic imaging, a hematopoietic cancer, including multiple myeloma, leukemia, or lymphoma. n various embodiments, the present teachings include use of an. adenovirus vector comprising a ROB04 enhancer/promoter operatively linked to a transgene for the manufacture of a medicament to treat a disease such as, without limitation, a benign disease, such as, without limitation, an inflammatory disease such as rheumatoid arthritis, atherosclerosis, psoriasis, Crohn's disease, ulcerative colitis. Type 1 (juvenile onset) or diabetes. In various embodiments, the present teachings include use ^: f an adeno virus vector comprising a ROB04 enhancer/promoter operatively linked to a transgene for the manufacture of a medicament to treat a disease such as, without limitation, an inflammatory and degenerative central nervous system disease such as Alzheimer's disease, multiple sclerosis, Parkinson's disease or amyotrophic lateral sclerosis, in various embodiments, the present teachings include u e of an adenovirus vector comprising a ROB04 enhancer/proaioter operatively linked to a transgene for the manufacture of a medicament io treat a disease such as, without limitation, osteoporosis via endothelial angiocrioc osteoclast inhibition alone or combined with concomitant angioetioe osteoblast stimulation. In various embodiments, the present teach ings include use of an adeno v irus vector comprisin a ROB04 enhancer/promoter operatively linked to a transgene for the manufacture of medicament, to treat a disease such, as, without limitation, a vascular insurllciency/iscbemic disease such as coronary artery disease, lower limb arteriosclerotic vascular insufficiency (peripheral vascular disease), or ischemic stroke. In various embodiments, the present teachings include use of an adenovirus vector comprising a OB04 enhancer/promoter operatively linked to a transgene for the manufacture of a medicament to treat a disease such as, without limitation, a C S disease such as cerebral vasospasm following subarachnoid hemorrhage.

Brief Descript ion of the Drawings

FIGS. 1A-1B illustrate Ad OB04-EOFP expression, showing upregulation. of endogenous ROB04 io orthotopic and xenograft tumors, FIG. 1 A illustrates irnrmmobiots of liver (Li), kidney orthotopic (KG) and subcutaneous (SCj xenograft tumors derived from 786-0 renal cell carcinoma cells probed with a polyclonal ROB04 antibody. FIG. IB illustrate densitometry of RO.B04 protein expression normalized to the endotheiiai cell (EC) marker VE-Cadherin reveals induction in tumors from both locales.

FIGS . 2A-2C illustrates vascular restricted RO.B04-di.rected reporter expression in kidney orihograft and subcutaneous heterograit tumors. Injection of .1 ,5 10' ¹ Ad5ROB04-EGFl^> (ROB04) or Ad5CMV~BGFP (C V) viral particles (vp) in hCAR transgenic;Rag2 O/ O mice produces extensive and intense reporter gene expression localized to microvessel endothelial cells in FIG. 2A subcapsular kidney orthotopic (KO), and in FIG. 28

subcutaneous (SC) xenografts. FIG. 2C illustrates imraunoblots and densitometry .normalized to either b-tubulio or VE-Cadheria reveal elevated HGFP reporter protein expression in both types of tumor. "K'\ host kidney, arrow, glomerular tufts, arrowheads and "T" mark tumor boundaries in left panels whereas arrowheads indicate endotheiiai tip cells in right upper ROB04 panels in PIG, 2A and FIG. 2B. Magnifications: 40X and 2 X; Red,

endomuein CD3! cocktail; Green, EGFP imraun.0 fluorescence. In FIG, 2 and subsequent drawings based on multi-color originals, gray-scale versions of each, color channel (red, green and blue) are shown, as well as a composite gray scale that combines all 3 (RGB) color channel?, in each case, the top left panel i the red channel, the top right panel is the blue channel, the bottom, left panel is the green channel, and the bottom right channel is the composite.

FIGS. 3Ά-3Ό illustrate that Ad5ROB04 can transcriptionally target metastatic endothelium. R.OB04-directed expression also differentially detected in circumferential microvessels immediately adjacent to ovarian follicles, asterisks in FIG. 3 A and FIG . 3B, but not in stromal microvessels, Ad5ROB04-directed expression is also evident in most mkrovessels within a peritoneal 786-0 renal cancer metastasis compared to nearly undetectable expression in adjacent host fallopian tube mkrovessels, asterisks, (FIG. 3D). Magnification of FIG. 3A and FIG. 3B 40X, FIG. 3C 200X, FIG. 3D 100X. Red, endomue i/CD31 cocktail; Green, EG.FP immunofluorescence in FfGS. 3A„ 3C, and 3D. FIG. 3B EGFP immunohistochemistr and hematoxylin counterstain.

FIG. 4 illustrates Ad5 vector expression in a host organ panel in tumor bearing

hCA :Rag2KO O composite mice, Magnification: 1 GX; ed, endom cin/CD3 ί cocktail; Green, EG F 1 ί nimitiiotluorescence ,

FIGS. 5A-5B illustrate that warfarin pretreatment detargets liver sequestration. FIG. 5A illustrates widespread high-level hepatoeyte EGFP expression in tumor bearing Rag2 O/KO mice injected with 1x10" vp Ad5CMV-EGFP. FIG. 5B illustrates warfarin pretreatment, 5 nig/kg, on day -3 and - i prior to vector injection at day 0 markedly decreases the frequency of hepatoeyte EGFP expression. Warfarin preheated detargets liver sequestration. Red;

CDS i/endoraueiii, Green; EGFP, Blue; DAPI. A. 10X, B. 200X.

FIGS. 6A-6.B illustrate warfarin liver detargehng in Rag2KO/KO mice increases the endothelial specificity of the Ad5ROB04 compared to the Ad5CMV vector. Fig. 6 A illustrates injection of 1x10^U vp of AdSROB04~EGFP into mice sans the hCAR transgene essentially abrogates endothelial expression in all organs except for liver and spleen, FIG. 6B illustrates irnmunohlotting corroborates trace detectable host organ EGFP protein expression in all host organs except for liver and spleen, W(-): vehicle injected mice, W{ ): mice treated with day -3/-I warfarin prior to vector injection. Magnification lOilX; Red, endomuem TB ! cocktail; Green, EGFP,

FIGS. 7A-7C illustrate warfarin liver deiargetiiig enhances tumor neovascular endothelial cell specificity of the A45ROB04 vector. The Ad5ROB04 vector mediated sporadic but easily detectable tumor endothelial cell EGFP immunofluorescence in both FIG, 7 A orthotopic and FIG. ?B subcutaneous 786-0 tumors grown in vehicle-treated Rag2KO/KO mice. FfG. 7C illustrates that EGFP immunobloiting and densitometry reveal warfarin-mediated reporter expression in both tumor types concomitant with decreased liver expression. Arrowheads: lutnor-kidney boundary, rectangle; area of low power image detailed in adjacent panel.

Magnifications: 40 and I 0X; Red, endoraucin/CD31 cocktail; Green, EGFP.

FIGS. 8A-8B illustrate Ad5ROB04 retargets liver expression to hepatic ECs following IV injection. FIG. 8A illustrates AdSCMVEGFP, FIG. SB illustrates Ad5 ()B()4- EGFP.

Red Greea'Blue as abo ve. Magnification FIG . 8A 100X, FIG. 8B 200X. An embodiment of the EC targeted Ad vector can detargei the liver for transgene expression.

FIGS. 9A-9C illustrate Ad5 OB0 tumor EC expression. Red: CD31/e«domucm,

Green: EGFP, Blue; DAFT. FIG. 9A illustrates subcutaneous 786-0 tumor, FIG, 9B and FIG.

9C illustrate "Krukenberg" intra-ovarian 786-0 metastases. Arrowheads: tumors. Asterisks host ovarian follicles. Red/Green/Blue as above, Magnification FIG. . and FIG. 9B !OftX,

FiG. 9C 200X. Tumor EC expression bias produces widespread intratumoral EC expression.

FIG. .10 illustrates Ad5ROB04~EGFP intra-, and peri-linxioral marrow expression in an IGR-

CaPl tibial metastasis. Green line outlines proximal tibial tumor. Red: CD3 i/endomuci

Green; EGFP, Blue; DAPI. 100.X.

FIG. I I illustrates EC angiocrine-targeted Ad vector strategy.

FIG. 12 iilustraies .femur BM from a CXCL12-GFP mouse, investment of bone sinusoidal vascular ECs by CAR-F.GFP cells. The ECs (Red) are ensheaihed. by CXC12- Abundant Reticular (CAR) cells (Green). Blue; DAPI. 400X.

FIG. 13 illustrates an embodiment of an EC targeted ptodrug-converting enzyme Ad vector AdSROB04-bCDD314A.

FIG. 1.4 illustrates vector and dose specific toxicity.

FIG. 15 illustrates focal bone marrow ablation mediated by Ad5ROB04-bCD production of 5-FU following 5-FC 500 mg kg BID IP. Red; CD3 t/eadomucm, Green; EGFP, Blue; DAPI. lOOX,

FiG. 16 illustrates lineage .reporter transgenic mice.

FIG, .17 illustrates strategy for simultaneous quiescence testing of PC A CSCs and host stem cells.

FIG. 18 illustrates metastatic implantation inhibition by liver targeted the AdCMV- SCXCR4/SDF 1 ligand decoy.

FIG. 19 illustrates a diagram of an embodiment of an EC-targeted Ad5 SDF1 ligand decoy. FIG. 20 illustrates Ad5ROBO-sCXCR4 mediated blood and spleen hematopoietic mobilization in C57mice. B: blood, S: spleen, BM: bone marrow.

FIG. 2.1 illustrates strategy for NOTCH/ WNT path ay activation. FIG. 22 illustrates polydstronk e-DNA. for creation of a gutless, "t¾era«ostic". Ad vector embodiment

FIGS. 23 -23C illustrate incorporation of MBP into Ad5 increased, viral gene expression to vascular beds of multiple host organs, FIG. 23 A illustrates immunofluorescence microscopy analysis of vector EGFP expression in host organs following intravenous injection of 0^iJ viral particles (vp) of Ad.MBP.CMV into adult C57BL/6J mice. FIG. 23B illustrates EGFP fluorescence per ηι² of tissue section area (FL fluorescence intensity) in each organ derived from AdS.CMV^'-snjected mice (n--4 for ail organs) versus thai from AdMBP.CMV-injected mice (n" I Q for liver, spleen, heart, kidney, muscle, small bowel, and brain; n^™? for lung, pancreas, and large bowel). FIG. 23C illustrates the percentage of vascular EC area expressing EGFP in each organ derived from AdS.CMV-injeeted mice {n--4 for all organs) versus that from Ad.MBP.CMV-injected mice (n^^'lO for heart, kidney, muscle, small bowel, and brain; n~7 for lung, pancreas, and large bowel). Bar graph is mean */- standard deviation asterisk: adjusted p<0.05, Magnification.: 100X, Red: endomucin/CD31 , Green: EGFP immunofluorescence,. Blue: DAPL Li; liver, S: spleen, Lu: lung, H: heart, K kidney, M: muscle, P: pancreas, SB: small bowel, LB: large bowel, B; brain.

FIGS. 24A-24B illustrate that warfarin pretreatmem reduced. Ad.MBP.CMV liver tropism but did not alter gene expression, m ther host organs.. FIG. 24 A illustrates warfarin, 5 rag/kg, on day ~3 and -1 before vector injection diminished hepatocyte expression hut did not change transgene expression i spleen. FIG. 24B illustrates EGFP fluorescence per μη of tissue area in each organ derived from warfarin-treated mice (n^;;;3 for all organs) normalized as percentage of the mean value of vehicle-treated or untreated counterparts (n~ 10 for liver, spleen, heart, kidney, muscle, small bowel, and brain; n^¾7 for lung) with standard deviation. Spleen (S), lung (Lu), heart (H), kidney (K), muscle (M), small bowel (SB), or brain (B). Asterisk indicates adjusted p<0.05. Magnification: I00X, Red: CDS l/endomucin, Green: EGFP immunofluorescence, Blue: DAPL

FIGS. 25A-25C illustrate that systemic administration of a low dose of Ad.MBi\CMV into adult mice produced differential and. non-linear reduction in gene expression in host organs. FIG. 25A illustrates EGFP expression in host liver, spleen, lung, and brain following intravenous injection of IxlOⁿ or 2x10¹⁰ vp of Ad.MBP.CM^'V into adult mice. FIG. 25B illustrates EGFP fluorescence per urn² of tissue area In eacli organ derived from the low-dose group (n-"6 for each organ). FIG. 25C illustrates rtormalization of the tissue EGFP fluorescence intensity values in FIG. 25B to the mean value of the high-dose counterparts. Asterisk indicates p<0.05. Magnification: S OOX, Red: endomucm CD31, Green: EGFP immunofluorescence,. Blue: DAPL Li: liver, S. spleen, Lu: lung, H: heart, K: kidney, M: muscle, P: pancreas, SB: small bowel B: brain.

FIGS. 26A-26B illustrate that depletion of circulating monocytes and hepatic and splenic macrophages lead io an increased Ad.MBP.CMV gene expression in the lung without a significant change in gene expression in other organs. FIG. 26A illustrates representative flow cytometry plots (left panel) quantifying tin* FSC-high SSC-low/CDT 1 b«positi ve/C 45- positive monocyte population in circulation. FIG. 26B illustrates EGFP fluorescence per i r of tissue area in each organ deri ved from the saline-injected mice (w* 7 for liver, spleen, heart, kidney, muscle, pancreas, small bowel, and brain; u^∞4 for lung) versus c!odronate liposome- injected mice (n^::::8 for liver, spleen, heart, kidney, pancreas, small bowel, and brain; n--7 for muscle; 5 for lung). Liver (Li), spleen ($), heart (fl), kidney ( ), muscle (M), pancreas (P), small bowel (SB), or brain (B). Asterisk indicates adjusted p<0.05.

FIGS. 27A-27B illustrate Ad.M^'BP.ROB04 detargeted hepatoeyte expression but reduced .levels of vascular EC expression in other host organs. PIG. 27A. illustrates EG P expression following intravenous injection of .1x 10' ¹ vp of Ad.M.BP.R.OB04 into adult mice. FIG. 27B illustrates the BGFP -positive vascular area analysis was performed as shown in FIG, 23C. Magnification: I OX, Red: endomucin/CD3t , Green.: EGFP immunofluorescence, Blue: DAP.I, Li: liver. S: spleen, L.u: lung, R: heart, : kidney, M: muscle, SB: small bowel, LB: large bowel, B: brain,

FIGS, 28A-28B illustrate that incorporation o MBP into AdS deiargeted the virus from liver hepatocytes, modestly increased gene expression in splenic marginal z e, and markedly enhanced gene expression in all regions of the brain. FIG, 2SA illustrates EGFP expression in liver and spleen following intravenous injection of 1x10" vp of AdS.CMV or Ad.MBP.CMV into adult C57BL 6J mice, FIG, 2 B illustrates immunofluorescence microscopy analysis of EGFP expression in. different regions of the brain following intra venous injection of 1 10^{s !} vp of Ad.MBP.CMV into adult C57BL/6J mice. Magnification; 100X, Red:

endoniucin/CD31. , Green: EGFP immunofluorescence. Blue: DAPL

FIG. 29 illustrates that Ad.M BP.CM V selectively targeted vascular ECs but not pericytes in multiple host organs. High-power magnification ΕΡί. micrographs of tissue sections co- stained with an endomucm/CD31 cocktail (top _.panels) and an EGFP antibody localized Ad.MBP.CMY transgene expression to vascuiar ECs by the in lung, heart, kidney, muscle, small bowel, large bowel, and brain. Magnification: 400X, Red: CD3J./endomucin for top- row panels, PIX FRp for middie-row panels, and NG2 for bottom-row panels. Green: EGFP immunofluorescence,. Blue: DAPL

1.9 FIG. 30 illustrates Ad.M8P.C V targeted cell population(s) distinct from CD45~pos.ittve or F4/8G-positive ceils in most host organs. Magnification: 400X, Red: CD45 for top-row panels and F4/80 for bottom -row panels. Green: EGFP immunofluorescence. Blue: DA PI.

FIG, 3.1 illustrates depletion of hepatic and splenic macrophages by ciodronate liposomes. Micrographs show F4/80 expression in liver and spleen from saline-treated mice (veh) or ciodronate liposome-treated mice (clod). Magnification: 10ΌΧ, Red: P4/80, blue: DAPI, FIGS. 32A-32F illustrate induced expression f Ad.MBP, R0BO4~EGFP and

Ad.RGD.ROB04-EGFP vectors in region of ischemia-reperfusion 0/R) in a suture mouse model FIG, 32 A illustrates Ad.MBP,ROB04 expression in the left ventricular l/R region. FIG. 328 illustrates Ad.MBP.R0804 expression in left ventricular septum. FIG. 32C illustrates Ad.MBP..ROB04 expression in right ventricular free wall. FIG. 32D illustrates Ad.RGD.ft0BO4 expression in left ventricular IM region.. FIG. 32E illustrates

Ad.RGD.R0B04 expression in left ventricular septum. FIG, 32F illustrates

Ad.R.GD.ROB04 expression in right ventricular free wall. Red: vascular endothelial specific immunofluorescence using a CD3 l/endomucin antibody cocktail Green: EGFP

immunofluorescence. Blue: DAPi nuclear stain. Magnification; 40X.

FIG. 33 illustrates Ad.MBP.ROB04.EGFP expression in the vascular endothelium of the adductor (thigh) muscle following hmdlirob ischemia secondary to femoral artery ligation. Red, Green, Blue as in FIG. 32. Mag: 40X.

FIGS, 34A-34C illustrate adenoviral vector expression localized within angiogenic villi in a small, bowel resection (SBR) model. FIG. 34A illustrates mice injected with

Ad.MBP.ROB04-EGFP five days post sham surgery. FIG. 34 B illustrates endothelial and possible lymphatic expression of the same vector in. angiogenic villi post SBR. FIG. 34C illustrates high power view of villous in FIG, 34B (arrowhead} showing coloealize vector transgene expression in angiogenic sprouting endothelium (arrowheads indicate sprouts). FIG, 34A and FIG, 34B l OOX, FIG, 34C 400X,

FIG, 35 illustrates Ad.MBP.CMV vector expression to the vascular endothelium surrounding the hypothalamus (encircled). Red, Green, Blue as in FIG, 32. Mag; 40X.

FIGS. 36A-36C illustrate expression of Aci.RGD. il5/FI3 vector within the vascular endothelium of human prostate brain metastases in a mouse. FIG, 36Ά illustrates a histological section that is adjacent to FIG, 36B. FIG. 36C illustrates a prostate brain metastases in another mouse. Asterisks denote metastases, cross uninvolved brain. Red, Green, Blue as in. FIG. 32. Mag: 100X. FIGS, 37A-37B illustrate Ad.RGDJ- /H3.ROB04 vector expression in bone marrow sinusoidal endothelium. FIG. 37A illustrates cortical bone marrow in bone shaft. FIG, 37B illustrates trabecular bone marrow near bone end and cartilaginous plate. Red, Green, Blue as in FIG. 32. Mag; l OO

FIGS. 38A-38B illustrate expression of A&RGD.RGB04-EGFP m a IGR-CaPl human prostate cancer femoral bone metastases in NOD/SClD/BL2Ry immuaodefieient mouse. FIG. 38 A illustrates an adjacent section to FIG. 38S. Gree and yellow asterisks are hematopoietic cells adjacent to metastasis. White and black asterisks are de novo, osteoblastic bone. White and black crosses are metastatic ceils. Arrowhead delineates osteoblastic "rimming", a pathological hallmark of osteoblastic metastases. Red, Green, Blue as in FIG. 32. Mag: J OO . FIGS. 3 A-3 D illustrate angtocrine production of 5~i1tsorouracii (5-FU) from bone marrow sinusoidal endothelial cells expressing cytosine deaminase (bCD) from an Ad.ROBC4 vector. FIGS. 3 A-39D illustrate bone trabecular histology from a mouse injected with Ad. ROB0 - EGFP control virus. FIG. 39.8 illustrates corresponding vascular marker

immunofluorescence, FIG. 3 C illustrates bone trabecular histopathology S-FC treated mice following Ad..ROB04~bCD and preinjection warfarin to detarget liver hepatocyte vector sequestration. FIG. 39D illustrates vascular immunofluorescence demonstrating dilated but intact, vasculature and apoptotic hematopoietic cells. Red and Blue as in FIG. 32. Mag: 100X.. Detailed Description

Abbreviations

Ad adenoviral/adenovirus

Ad5 adenovirus serotype 5

ANOYA analysis of variance

BM bone marrow

CAR Coxsaekie and adenovirus receptor

CMV cytomegalovirus

CSC cancer stem cell

DAPl. 4-'_>6-diamidino^>2-plienylindole

EC endothelial cell

EGFP enhanced green -fluorescent protein

GFP green fluorescent protein

HPC hematopoietic progenitor ceil

KO knock-out or kidney orthotopic

PCA prostate cancer PVDF fx>iy vinyl idene difmoride

RCC renal cell cancer

ROB04 Magic Roundabout

SC subcutaneous

VE-Cadherin vascular endothelial cadherin

VEGF vascular endothelial growth factor

vp viral particles

The present inventors have found that an angiocrine niche can affect angiogenic inhibitor resistance, and can provide a focal mieroeiwironment for selection of aggressi ve tumor emergence. They thus modified vascular endothelial angtocrine functions for malignant and benign disease treatment using endothelial targeted adenoviral vectors.

The present inventors exploited the intact vasculature and the endothelial cells cootasned therein as a vehicle for delivery of therapeutic agents in benign and malignant disease. The vasculature can provide access to diseased tissue and the vascular endothelial cells are in close approximation of target ceils within diseased, tissue which allows for increased and more specific targeted dosing of therapeutic agents. The vascular endothelium is the first cell type/organ encountered, by adenoviral vectors. Thus, systemic intravenous or intraarterial, vector injection, can target vascular endothelium first prior to uptake in.

nonvascular cells in organs and tissues.

in some configurations, an endothelial targeted adenoviral vector can be modified for cargo gene expression that is restricted to disease tissue microenvsronments. The

mieroenvironment can include different cell types in addition to the diseased cells. These cell types can include but are not limited to ancillary cell types including fibroblasts,

inflammatory cells- myeloid cells, macrophages and lymphocytes, and fibroblasts.

Collectively the crosstalk between diseased cells and the ancillary cellular collection can alter the tissue microenvironrneni These alterations can include but are not limited to low oxygen, low pH- high acidity, altered redox potential, and intracellular stress, in some embodiments DNA regulatory regions-enhancer/promoters that are solely activated by one or more diseased tissue microenvironmental alterations can foe employed- These enhancer/promoters can be engineered into adenoviral vectors to increase diseased compared to normal tissue specificity.

Diseases and/or conditions to which endotheiial-targeted adenoviral vectors of the present teachings can be applied can include but are not limited to cancers, such as without limitation solid organ primary site (site of origin) cancer, brain cancer, solid organ metastatic cancer including font not limited to bone, lung, liver, and lymp nodes, occult cancer metastatic imaging, hematopoietic cancers including but not limited to multiple myeloma, leukemia, and lymphoma; benign diseases: inflammatory diseases including but not limited to rheumatoid arthritis, atherosclerosis, psoriasis, Crohn's disease, ulcerative colitis, juvenile onset diabetes and Type 1 diabetes, inflammatory and degenerative central nervous system diseases including but not limited to Alzheimer's disease, multiple sclerosis. Parkinson's disease, and amyotrophic lateral sclerosis, osteoporosis via endothelial angiocrine osteoclast inhibition alone or combined with concomitant angiocrine osteoblast stimulation, vascular insnfficiency/ischemic disease including but not limited to: coronary artery disease, lower limb arteriosclerotic vascular insufficiency (peripheral vascular disease), and ischemic stroke, and other central nervous system diseases including but not limited to cerebral vasospasm following subarachnoid hemorrhage

in addition to perfusion for nutrient and oxygen provision, endothelial cells (ECs) ears produce and secrete growth factors, ehemo- and cy tokines into their local nucroenvironment. This EC function can regulate other stromal cells such as fibroblasts, inflammatory cells, organ parenchymal cells. ECs can regulate adjacent cells by "appositiona!" signaling that includes direct attachment of adjacent cells to the abluminal EC surface and engagement of membrane tethered growth factors, receptors, and: other EC surface molecules that interact with receptors on the adjacent stromal and organ parenchymal cells. Cancer or benign ceils (in particular cancer or organ stem cells) can also be regulated by these EC angiocrine functions.

Embodiments of the present teachings include the structure and use of adenoviral vectors carrying transgenes. Configurations can include adenoviral vectors that can selectively enter (transduce) and/or can foe exclusively expressed in vascular ECs, in some embodiments, a vector transgene can encode a prodrug-converting enzyme. An expressed enzyme can produce an acti e cytotoxic chemotherapy drug following inactive prodrug administration., in other embodiments, a transgene can generate, or prodrugs can elaborate conversion product molecules that are secreted, by ECs into the tissue mieroenvironments, in other embodiments, a transgene can be expressed in ECs and activate EC surface molecules which can affect cellular function in an adjacent microenvironment In some embodiments, for benign diseases, a vector transgene can encode a molecule that can inhibit inflammation by sequestration of ehemo- or cytokines, in some embodiments, a vector transgene can encode a molecule that can stimulate disaggregation of plaque formation in Alzheimer's disease. In some embodiments, adenoviral vectors can be engineered for EC-specific vector entry (transducttonal targeting) and/or they can be engineered to contain a DMA

enhancer/promoter ( A regulatory element) that can be specifically expressed in EC's. In other embodiments, adenoviral vectors can be engineered with transgenes that cats include but are not limited to promoter independent regulatory elements including raiero NA seed sequences. 3 ' m A stability elements, and/or mRNA elements containing mR A

secondary structure that can be translated in microenvimnmentally stressed states such as hypoxia or altered redox.

Some embodiments of the present teachings include vector-mediated, subversion of endothelial cell (EC) angiocrtne functions, which can he used to "cripple" host niche ceils that surround ECs and closely appose cancer stem cells fCSCs). In some configurations, the vasculature can be preserved and can redirect ECs to produce secreted molecules in order to dysregulate CSC niche sites throughout bone metastases. in some embodiments, EC-targeted Ad vector configurations Can detarget the liver for transgene expression (FIG. 8). In some embodiments, tumor EC expression of the vecior configuration bias can produce widespread robust intraturooral EC expression (FIG. 9),

n some configurations, an IGR-CaPi prostate cancer cell line derived from a Gleason grade ? radical prostatectomy can gro as gland-torming adenocarcinomas, and form, mixed osteoblastie/osteolytic bone metastases in (immunodeficient) mice (A! Nakouzi ai. 2012), These IGR-CaPi cells can be androgen independent, and can be enriched for PCA CSC markers (Chauchereau et l 201 1 ), In some aspects, .EC-targeted Ad vector configurations can be expressed within and adjacent to IGR-CaPl bone metastases (FIG. 10).

in some embodiments, an EC-targeted Ad vector of the present, teachings can dysregulate peri vascular bone niches which can be essential tor CSC maintenance, in some configurations, an Ad. vector of the present teachings can contra.! metastatic growth either via enforced CSC differentiation, or b chemo/irradiation therapy synergism due to .proliferative transit amplifying cell population expansion, in some embodiments, an Ad vector of the present teachings in combination with bone niche lineage tracing, cell cycle quiescence, and stem cell ligand signaling reporters, can be used to elucidate PCA CSC hone niche dynamics, in some embodiments, angiocrine-targeted Ad vectors can translationaily transition to clinical therapeutics.

In some embodiments, the present teachings include use of tumor blood vessels to access the most remote regions of tumor. In some embodiments, the present teachings include hijacking the perfusion independent "angiocrine" vascular EC functions to produce active drug metabolites or secrete CSC ligand decoys locally and at high, levels within bone marrow CSC metastatic niches, in some aspects, ibis approach can be performed by commandeering EC angiocrme functions using Ad vectors with a predominant metastatic neovascular expression (FIG. 9, 10). In various configurations, this approach can allow for prodrug end product elaboration specifically within metastatic niches for the elimination of systemic toxicities such as stomatitis, diarrhea, or heart failure typical of sy stemic chemotherapy. In some configurations, an EC-targeted Ad vector of the present teachings can be used to preserve and/or exploit the mtratumoral vasculature while avoiding multiple tumor cell autonomous and microenviromnenta! alterations.

In some embodiments, the present teachings include EC angiocrine secretion modulated by a modified Ad vector {FIG. 1 3) for targeting metastatic cancer. Ad vector- mediated exploitive, engineering of EC angiocrine secretion is a therapeutic strategy for targeting metastatic cancer. (FIG. 11 ) Metastatic cancer can include, without limitation, prostate cancer, which, can metastasize to the hone. In some aspects, multiple niche cellular components within the bone marrow can be targeted, in some aspects, EC targeted Ad vectors of the present teachings can be expressed at high levels in BM. sinusoidal ECs both within and adjacent t osteoblastic PCA metastases {FIG. 10). in some aspects, there can be perivascular apposition of niche cellular components combined with hone sinusoidal capillary fenestrations, hi some aspects, vectors of the present teachings can be used to dysreguiate and disrupt bone PCA CSC niches. In some aspects, one ECs can be targeted tor expression of the 5 -fliioro uracil (5FU) prodrug converting enzyme, cytosine deaminase.

In some embodiments of the present teachings, an aiigiocrine-engineered Ad vector thai expresses a stem cell ligand decoy can be used to differentially mobilize PCA CSCs from metastatic bone niches, in some configurations, the C CR4-SD1^" I axis can be disrupted through, expression of a decoy such as, without limitation, a truncated NOTCH or WNT ligand decoy.

in some configurations, PCA CSC mobilization effectors can. be selected to test combinatorial enhancement of the PCA standard of care ehemoinerapeutic, doeetaxel, in some configurations, Ad-sNOTCFI. and Ad-sF Z{ WNT) ligand decoys in cell culture can be constructed and functionally tested. Combinations of vector embodiments for additive or synergistic PCA CSC mobilization can also be tested., in some configurations, "gutless" poiycisironic Ad vectors ligand decoy(s) can be constructed. Such a polycistron can. be under switchabie control and can obviate constitutive low-level host stem cell mobilization and can. provide the potential for a synchronous prodrag-medsated cytotoxic therapy combination. A LUCrGFP fusion construct can be included. In some configurations, a polycisironic vector configurations can be tested in a bone metastatic mode!. In some aspects, a gutless vector can persist for a prolonged period following a single systemic administration, and can elicit minimal preformed immune responses. In some aspects, a large gutless vector configuration transgene capacity can offer theranostic potential for combining therapeutic and imaging capabilities into one vector embodiment.

Some configurations include Ad vectors with EC specific expression (FIG, 8, FIG. 9, FIG. 10), in some aspects, these modified vectors can include a 3 kb ROB04

enhancer/promoter (Okada ei ah 2007). The OB04 enhancer/pronjoter fragment, can include multiple ETS and hypoxia-inducible factor hypoxia response elements (Okada et al. 2007; Okada el al. 2008). These elements can impart an expression bias for intra- and peritumoral vasculature (FIG. 9., FIG. 10). Most of the AdROB04 vector can be sequestered in the liver (FIG. 5) (Waddington et al. 2008), Liver sequestration can be predominantly mediated via coagulation factor binding to the adenovirus capsid (Waddington et al. 2008), liver-detargeting efficacy of warfarin pretreatment in mouse models (FIG, 5) can be validated. The AdCMV vector configuration was used to visualize hepatocyte reporter expression. 786-0 renal cell carcinoma ( CC) cells were used. There was an induction of AdROB04-EGFP expression in primary xenograft and metastatic ECs (FIG. 9 and FIG. 10)., In contrast, host organ expression of the AdSROB04 vector was restricted to scattered ECs within liver and spleen. Western blotting and densitometry normalized to the EC-specific VB-Cadherin revealed that Ad5 ROB04 reporter expression was greater in tumor versus liver (FIG, ^'! ). Fiver detargeted EC targeted Ad vector configurations can be used for therapeutic purposes (Short et ai 201 ).

Is some configurations, PCA ceil lines such as PC3, and LNCaP as PCA models can be tested. Data reveal exquisite EC troptsm of MBP vector embodiments in the vasculature of several host organs. The CMV promoter used in these experiments mediated this host organ EC expression. The MBP vector has EC specificity conferred by vector entry (transduction).

In some embodiments, Ad vectors can be tailored for enhanced or restricted tumor EC specificity by choosing from a menu of promoters solely or preferentially activated by the tumor .microenvironment These include but are not limited to promoters activated by hypoxia (Heidenreich ei at. 2000; Greenberger et i. 2004; Marignol al. 2009), DMA. damage (Eeonoraopoalou et al. 2009: Westerink ef al, 2010), or endoplasmic reticulum stress (Zetig et al. 2009; He et al. 2010), ail of which are induced in. ECs within tumors. In some embodiments, Ad OB04 can be shown to direct expression in three host organs (liver, spleen, and bone marrow). In various embodiments, PCA bone metastases elicited a peritumoral recruitment of Ad vector expressing ECs. OB04 can achieve sufficient bone metastatic specificity.

ECs are niche components. ECs are the source of secreted growth factors, cheraokme ligands, and membrane tethered molecules thai maintain CSC persistence. This short range signaling has been designated as "angiocrine" functions. The present inventors have created EC targeted Ad vector configurations that have angioerine activity, including in bone.

Aagtocrine-targeted Ad vectors can be used to achieve metastatic growth control via CSC depletion either alone or in combination with cytotoxic therapies, in some configurations. Multifunctional "iheranosik" Ad vectors can he created with trans! atkmai applicability.

In various embodiments, promoter and promoter fragments can be utilized in the Ad vector embodiments for target functions. Tumors, including bone metastases, can be hypoxic. Promoter ^'fragments from. VEGF or endothelin; both of which contain hypoxia response elements cognate can thus be used for hypoxia-inducible factor- ! and -2 (Heidenreieh et al. 2000; Greenberger et aL 2004 ). In some embodiments, tumor vasculature can be under DNA damage stress (Economopoulou ef i. 2009). to some embodiments, the R.A 5 IC promoter upstream of major DNA repair enzyme can he used to induce for D A repair ( Westerink el al. 2010).

in some aspects, tumor vessels ca also be under endoplasmic reticulum stress

(unfolded protein responseUPR) {Zeng et al. 2009).. in some aspects, the cognate promoter for the XBP 1 transcription factor whose alternative splicing is only induced during U.PR induction (lie et al 201 ) can be used. In some aspects, tumor vascular specificity can be increased with the Ad5R.OB04 vector, in some aspects, roicro- ^'NA seed elements can be placed in. the UTR of the CD or any other cargo gene cDN^'A (Wang and Olson 2009).

In some configurations of the present teachings, there is a family of mi RN As that are dysreguiated in tumor aeovascuiature (Dews ei a 2006). These seed sites can promote cargo gene niR A degradation in quiescent host vessels and message stabilization in bone metastasis neovessels. There can also be other NA structural elements and tumor

microenvironnient-specifie interna! ribosome entry sites thai can be used, or inclusion of cD As encoding peptide elements targeting cargo proteins for degradation, in nonmoxic vessels or in host ECs not stressed by increased reactive oxygen species (Oikawa el aL 2012). These fusion proteins can be stabilized in bone metastatic EC^'s. An EC-targeted vector configuration, .BP~A.d5 (myeloid binding peptide) (Alberti et til, 20 ! 2), can be utilised to provide increased tumor specificity to separate therapeutic efficacy and host (Bfvl) toxicity. Engineering MBP display on the deknobbed AdS fiber shaft produces EC transduction (Alberti e i 2012). E and in vivo mouse experiments suggests thai myeloid cells presented the MBP vector to EC's. Our results show the EC iropis.ro of this vector configuration in the vasculature of several host organs. This host organ EC expression can be mediated by the CMV promoter, in some configurations, Ad vectors can be tailored for enhanced or restricted tumor EC specificity by choosing from a menu of promoters solely or preferentially activated by the tumor mieroenvironment. These can include promoters activated, by hypoxia (Heidenreieh et al. 2000: Greenberger et at. 2004; Marignol et i

2009), D A damage (Economopoulou et at. 2009; Westerink ei al. 201 0), or endoplasmic reticulum stress (Zeng et al. 2009; He et ai, 201. 0), all of which are induced in ECs within tumors. In some aspects, CSC mobilization potential and. cytotoxic chemotherapeutic- enhancement of our AdROBO-sCXCI SOFi iigand decoy ca n be utilized.

To demonstrate expression patterns of an Ad vector of the presen t teachings, a Ad vector containing 3 kb of the Magic Roundabout (ROB04) promoter transcriptionally regulating an enhanced green fluorescent protein (EGFP) reporter was injected into immunodeficient mice bearing 786-0 renal cell carcinoma xenografts and orthotopic tumors.

in some embodiments, the Ad5ROB04 vector, in conjunction with liver deiargeting, can provide genetic access for in-vivo EC genetic engineering in malignancies, AdSROBQ4- EGFP tumor EC expression was revealed in hCAR transgenic Rag2 knockout mice. In contrast, AdSCM V-EGf P was not expressed in tumor ECs.

As the hCAR. transgene also facilitated Ad5ROB0 and control AdSC V vector EC expression in multiple host organs, follow-on experiments engaged vvartarin-mediated liver vector detargeting in hCA.R negative mice. A.d5ROB0 - ediated EC expression was abrogated in most host organs, whi!e intra-tumqral neo vascular expression was spared.

in some embodiment, targeting tumor EC signaling pathways that encompass both angiocrine and perfusion functions can target the multi faceted resistance mechanisms of malignancies. Adenovirus (Ad) is a potential delivery vehicle for tumor EC targeting (Linderoann D ^'&ι al. 2009; Dong Z et at. 2009). Systemic injection of EC-targeted Ads can circumvent the challenge of tumor permeation vexing local vector injection, and. can address the challenge of diffuse, multiorgan, metastatic disease.

in some embodiments, endothelial targeting can be implemented using a

configuration of a first generation adenovirus serotype s (Ad5) vector. A transcriptional targeting strategy was engaged including creating a vector configuration whose reporter gene was regulated by the endothelial predominant Magic Roundabout (ROBQ4)

enhancer/promoter. In hypervaseular 786-0 renal carcinoma xenografts, orthotopic rumors, and spontaneous metastasis, Ad5ROB04 directed enhanced green fluorescein protein (EGFP) expression to the neovasculature, whereas a vector whose reporter was controlled by the human cytomegalovirus (CM V) enhancer/promoter failed to produce tumor neovascular reporter expression sufficient, for detection. Ad5ROB04 is a vector with the capacity for genetic- manipulation of tumor EC^'s to effect destruction or normalization of the malignant microeovironment.

ECs are one of. the primary cells exposed to intravenously injected particles. Tumor !Tiierovesseis are conduits that can facilitate imra-turnoral vector distribution particularly in hypervascular iiniwrs such as renal cancer metastases. Experiments were perforated on vector endothelial transcriptional targeting. A previousl characterized.3 kb enhancer/promoter of human ROB04 (Okada Y t al. 2007} was used to produce vascular endothelial, localized gene expression. In some embodiments, an AdS OBCM vector can be used to target the endothelium within primary and metastatic renal cancers, for example in in immunodeficient mice, in various embodiments, vectors and liver detargeted/ttmior EC retargeted, vectors can contribute to tumor EC-tailored gene therapeutics.

in some aspects, vector reporter gene expression can be quantified using quantitative imniunobloiting, with a combination of wide field Sow power and intermediate level microscopic magnification. The latter strategy can demonstrate evidence for vascular EC vector co-locaiixation within primary and metastatic cancers. Wide field imaging can be used to detect heterogeneous vector tumor vessel targeting. These results indicate that

combinations of vector configurations tuned to discrete microenvironments can be beneficial for efficacious tumor control.

In some embodiments, tumor nricroenvironment can selectively activate ECs for B04 expression, as demonstrated by endothelial transcri tional targeting using an Ad5 vector configuration with the ROB04 enhancer-promoter. An immunobiot analysis can provide evidence for endogenous E.OB04 induction in vascularized tumors compared to normal organs. Immunofluorescence data indicate that the tumor mtcroenvironment selectively activates ECs for ROB04 expression.

in some aspects, the 3 kb ROB04 enhancer/promoter fragment used in these studies was analyzed for elements crucial for endothelial specific expression. In some configurations, BTS family and Spl transcription factors can mediate endogenous gene induction for RO.B04 en ancer/proaioter fragment activity.

In some embodiments, the Ad5 OB04 caps id can be genetically manipulated to achieve liver detargetmg. Ad5ROB04 veefor-mediated tumor EC expression can be

demonstrated following factor X-mediated liver deiargeting. Our data demonstrate methods of exploitation of CAR independent vector transduction pathways in tumor EC's.

Vascular endothelium has been a sought after gene therapy target because of its immediacy to blood-borne therapeutics and its pathophysiological role in a wide range of benign and malignant diseases, (Dong, Z„ et al, 2009; Muro, S„ et al, 2004; Lindemano, P., et al. 2009; Aird, W.C., et ai. 200?) Despite their accessibility, vascular ECs are poor transduction targets for unmodified AdS vectors. (Baker, A M., et al. 2005} In addition, systernically administrated AdS is rapidly opsonized by circulating ig.Vi antibodies and complement components, leading to vtms clearance- by liver Kupffer ceils. (Duffy, M.R., et al. 20 s 2) AdS also avidly binds to blood coagulation factor X, which bridges the vims to hepatocytes by interacting with cell surface heparan sulfate proteoglycans. (Waddington, S.N., et al. 2008) Liver Kupffer cell clearance and hepatocyte transduction greatly limit circulating AdS vector efficacy. Thus, molecular engineering efforts to achieve Ad vector vascular targeting have focused on diminishing or abrogating liver tropism and opsonization while increasing EC transduction. (Duffy, MR.., et al 2012; aliberov, S.A., et al. 20 B) Over detargeting engaged genetic capsid modification. Virus opsonization diminution has been addressed using chemical shielding of AdS capsid proteins. (Duffy, M.R., et al. 2012} One approach to EC trarisductional targeting has been vector pseudo-typing. Ad vectors pseudoty ped with fibers or fiber knobs from different human, or from non-human, serotypes exhibited improved transduction efficiency of cultured human or rodent (rat ) ECs (Shinozaki, K., et al. 2006; Preuss, MA., et al. 2008; White, KM.., et al. 201 3). EC transduction has also been achieved through capsid fiber knob display of peptide ligands such as the argmine- glycine-asparate (RGD) motif cognate for the angiogenesis associated integrins th ps and α% , (Preuss, MA., et ai. 2008; ick in, S.A., et al. 2001 }. A parallel strategy for EC specificity has been transcriptional targeting using enhancer/promoter elements of

endothelial-specific genes such as VEGFR-2, VEGFR-l, preproemfathelm-J, and

roundabouts (Kaliberov, S.A., et l. 2013; Lu, Z.H., et al. 2013; Song, W., et ai. 2005; Greenberger, S., et ah 2004; Reynolds, P.M., et ai. 2001; Tal, R., et al. 2008). Transcriptional targeting restricts vector transgene expression to specific EC populations that in most instances are angiogenic and in some cases also, hypoxic. However, the transcriptional strategy, when applied alone_., does not alt r the K.upffe.r cell sequestration or hepatocyte transduction. Recent efforts have focused on the combination of transductional and transcriptional strategies to achieve^, enhanced organ or disease specific EC vector transgene expression. ( afiberov, S.A., et al. 2013; White, K.M.. et a!. 2013) Despite progress, systemic-ally administered Ad vectors are still ineffective in gene transfer to some clinically important organs. Dose escalation to achieve appreciable vector expression in marginally accessible organs likely will fail due to dose-limiting adverse effects such as liver toxicity, cytokine storm, or organ imperviousness to the vector. (Z-abs, A,K„ et al 2009) Collectively, the limitations of current EC targeting efforts reinforce the need for farther vector improvement.

Ad.MBP was previously shown to preserve the myeloid cell-binding specificity of the MBP peptide ex viva, ( Alberts, MX)., et al. 2012) but efficiently and preferentially target gene expression to the lung microvessel ECs in vivo, (Alberts, M.O., et ai. 2013) The latter work used single-ceil lung suspensions and confirmed that Ad.MBP solely bound to myeloid cells and not to ECs, Co-culture of virus-loaded myeloid cells on an EC monolayer provided indirect evidence supporting a myeloid cell-mediated viral "handoff ^* mechanism for potentiating the EC transduction. (Alberts, M.O., et al. 2013) Similar carrier cell hand-off or "hitchhiking" target cell transduction was proposed for other viruses in vivo. (Cole, C, et at 2CK⁾5; Roth, J.C., et al. 2008) A central tenet of vector hand-off postulates close contact of virus-carrier cells to the target cells enabling viral penton access with target cell iotegrins for internalization, bypassin the requirement of an initial attachment step in celi transduction. (Roth, J.C., et al. 2008) Indeed, the previous lung work revealed that the Ad.MBP virions rapidly bound to lung following intravenous injection. There the hypothesis was that vector attachment and "hand-off" to lung ECs was mediated by marginated neutrophils. (Albert!, M.O., et al. 201 3 } Our current data extend these findings and reveal that the MBP peptide possesses a much broader EC-specific trapism in vivo, and many of the permissive recipient organs exhibit a wide range of tissue-specific forms of resident myeloid cells. Our clodronate study provides evidence that circulating mononuclear cells and. tissue resident macrophages in liver and spleen are dispensable or redundant for mediating the Ad.MBP EC expression.

The Ad.MBP vector produces multi-organ vascular expression .following warfarin- mediated Factor X depletion, indeed, previous work- demonstrated that Factor X-virus hexon binding "shielded" the vector from peripheral natural antibody-mediated destruction in immunocompetent mice, (Xu, ,, et al. 2013) Multiorgan expression analysis also enabled us to discover the exquisite lung tropism of our Ad.MBP vector. Viral particle dose reduction essentially eliminated gene transfer to most organs while maintaining robust lung expression. This apparent pulmonary vascular avidity indicates that the Ad.MBP vector can be an ideal vehicle for treatment of pulmonary diseases, particularly (hose initiated by single gene mutations.

While Ad.MBP has many conceivable applications in other organs, its widespread expression in cardiac and brain vasculature is particularly exciting. In the heart, gene therapy has focused on ischemic disease (Tang, T., ei al. 2013), While the immediate cause of cardiac ischemia is coronary artery atherosclerosis, myocardial remodeling is the principal mechanism for development of chronic congestive heart failure (van Berlo, J.HL et ai 2013). Restoration of blood flow has been approached usin gene therapy as a surgical adjuvant or as primary treatment {Bradshaw, A.C., et at. 2013; arainsky, S.M, et ai. 2013), Our Ad.MBP vector can solve the dual challenge of coronary perfusion and myocardial

remodeling. Coronary perfusion can be increased using Ad.MBP vector armed with

constitutivety activ hypoxia-inducible factors ( iFs) Tal, R., et al. 2008). The widespread myocardial vascular distribution of Ad.MBP presents the opportunity to capitalize on EC angtocrme functions such that ECs are transformed into local sources of HiF-mediated angiogenic factors to both preserve marginal zone myocardial viability, and potentially augment arteriogenesis. Similarly, Ad.MBP vectors containing poiycistronic iransgenes encoding the same molecules apparently secreted by MSCs or ESCs could effect restorative rather than pathological myocardial remodeling by inducing expansion and myocardial differentiation of perivascular resident cardiac stern ceils (Katndar, F., et al. 2012; Ou, D,8„ et l. 2013),

Brain gene therapy strives to achieve long-term expression in neurological disorders such as Alzheimer's, amyotrophic lateral, sclerosis (ALS), or brain cancer (Conne, P.G., et at. 2012; Ramas aray, S., et al 201.2; Assi, .H._s et ai. 2012), The ability of our Ad.MBP vector to target greater than 62% of blood vessel beds in all regions of the brain offers the potential for treating the multifocal mtraparenchymal mechanisms for both diseases, in brain cancer, glioblastoma (GBM) in particular, the tropism of the Ad.MBP vector for brain vascular ECs can target peri vascular GBM stem cells by aogiocdne-mediated secretion of secreted cytotoxics or molecules blocking signaling pathways that maintain this therapy resistant cell population (Galan-Moya, E.M., et al 201 1 ; Zhu, T.S., et ai. 2 11).

The Ad.MBP vector enables unprecedented multi-orga vascular access. This vector can be used to harness ECs for production of a variety of therapeutic molecules for a diverse collection of benign and malignant diseases, its mul ti-organ tropism raay be uniquely beneficial, in cases wherein greater di sease specific sty is required, the inherent. EC vector tropism allows swapping in enhancer/promoters tailored to the altered raieroenvironment. created by each disease in each organ.

Materials and Methods

Methods and compositions described, herein utilize laboratory techniques well known to skilled artisans, and can be found, in laboratory manuals such as Sambrook, J., et al.„ Molecular Cloning; A Laboratory Manual 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001 ; Specior, D. L, et l.. Cells; A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, MY, 1998; Nagy, A., Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition). Cold Spring Harbor, NY. 2003 and Harlow, E., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. Methods of administration of pharmaceuticals and dosage regimes, can be determined according to standard principles of pharmacology well known skilled artisans, using methods provided by standard reference texts such as Remington: the Science and Practice of Pharmacy (Alfonso R. Gennaro ed. 19th ed. 1995); Rardman, J.G., e a!., Goodman & Oilman's The Pharmacological Basis of Therapeut ics, Ninth Edition, McGraw-Hill, 1 96; and owe, R.C , et al.. Handbook of Pharmaceutical Exeipients, Fourth Edition, Pharmaceutical Press, 2003. As used in the present description and any appended claims, the singular forms "a" "an" and "the" are intended to include the plural forms as well, unless the context indicates otherwise.

Animals

All mice were of C57BL 6J background and seven to fourteen weeks of age. Mice were obtained from Jackson Laboratory (Bar Harbor, ME) or through breeding in authors" animal facility. Experimental, procedures involving mice were carried out under protocols #201,20029 and #201 10035 approved by the Washington University Animal Studies Committee.

Cells and Adenovirus vectors

Human embryonic kidney HE 293 ceils were purchased from Microbix Biosystems (Ontario, Canada), Cells were cultured in DMEM/F I2 (Mediated), Hemdon, VA) media containing 3 % fetal bovine .serum (F.BS) (Summit Biotechnology, Fort Collins, CO), in a humidified atmosphere with 5% C02 at 37*C. Replication incompetent El - and ES-deleted AdS vectors were created using a rwo-piasmld rescue method. Plasmids encoded expression cassettes containing either the cytomegalovirus major immediate-early enhancer/promoter (CM V , or the human roundaboui4 (ROBCM) enhancer/promoter, each cloned upstream of enhanced green fluorescent protein. (EGFP) followed by the bovine growth hormone polyadenylarion signal. These expression cassettes were cloned into a shuttle plasratd

(pShuttle, Qbiogeoe, Carlsbad, CA) to generate the pShuttleCMV-EGFP and

pShuttleROB04-EGFP pfassntds, respectively, and Inserts were confirmed by using restriction enzyme mapping and partial sequence analysis. The shuttle plasmids were linearized with Pme I and integrated into the Ad.5 genome by homologous recombination with a pAd5 plasniid, encoding the native Ad5 fiber, or a pAdMBP plasratd, encoding an MBP-ftber-fibritin chimera, in the £. col t strain BJ5183. To rescue Ad. BP. OB04, the recombinant viral enome was linearized with Pac 1 and then transacted into 2 3F28 cells using SuperFect Transfection Reagent (Qiagen, Chatsworth, CA). 2 3F28 cells stably express the native Ad5 fiber; thus, viruses rescued at this point were mosaic in the sense that the Ad5 virions randomly incorporated a mixture of native Ad5 fibers and BP-fiherfibrstin chimeras. (Belousova, N., et al. 2003) After an additional, round, of amplification on 293F28 cells, the viruses were amplified in HEK293 cells, whic do not express native AdS fiber, to obtain virus particles containing only BP-fiber-fibritin proteins. The Ad.MBP.CMV vector containing a peptide sequence on a T4 ftbritm chimeric fiber knob was created as described previously. (Alherti, M.O., et al. 2013; Albert.!, M.O., et al. 2012) Recombinant viruses were purified by two rounds of CsCl density ultracentrifugation and diaiyzed in storage buffer containing 10 ramoI/L HEPES, I mmol/L MgCh, pH 7.8 With 10% glycerol as previously described. (He, T.C., et al. 1998} The viral particle (vp) concentration was determined by absorbance of dissociated virus at A260 run using a conversion factor of 1.1 x l(P

vp/absorbanee unit

Warfarin and ciodronate-liposome treatment

Mice were subcutaneously injected, with warfarin, 5 mg/kg in peanut oil, 72 hours and 24 hours prior to virus injection. (Short JJ,, et al, 2010} Clodronate-Iipososnes, 10 pL/g body weight, (CJodronateLiposomes.com- etherlands) or saline buffer were injected into the tail vein 48 and 24 hoars prior to vector injection, (van Rooijen, N„ et al, 2010} Twenty-four hours later, peripheral blood was collected by cheek pouch bleeding, and then Ad.MBP was injected.

Virus injection and host organ harvest

Mice were tail-vein injected with 1.x 10ⁿ or 2x 10^io particles of virus in 200 pL of saline. Seventy-two hours post virus administration, mice were anesthetized with 2.5% 2, 2, 2-txifc.romoelhanol (Avertin, Sigma- Afdrieh, St, Louis, MO.), perfused via the left ventricle with phosphate-buffered saline (PBS) followed by 10% neutral buffered formalin. Harvested organs were post-fixed in. lorraalin at room temperature tor 2 to 4 hours, cryo-preserved in 30% sucrose in PBS at 4^¾C overnight. Lung was further inflated and fixed by injecting formalin solution into trachea followed by closing the trachea by ligature and men processed as above. Treated tissues were embedded in NEG50 (Thermo Fisher Scientific, Waltham, MA) or ^"f issue-Tek OCT mounting medium (Sakura Torrance, CA, USA), and frozen in a liquid nitrogen pre-chilled, 2- methylbutane-contaming glass beaker.

Immunofluorescence staining

Ail mouse tissues were eryosectioned at 16 pm. Lung was also cut at 5 pm for determination of iransgene microvessel co-locaiimtion. Frozen section slides were air-dried for ten minutes, washed three times in PBS, blocked with protein block solution (5% donke serum and 0.1% Triton X-100 in PBS) for one hour, and incubated at 4°C overnight in protein block containing primary antibodies including: rat anti-endomucm 1 : 1 ,000, rat anti- PDGF p 1 :200 (#14-5851-81 , and #1.4- 1 02-8 1 , eBioscien.ee. San Diego, CA), Armenian hamster anii~CD31 1 : .1 ,000, rabbit anti-NG2 chondmitm sulfate proteoglycan. 1 : 100

(# ABf398Z and #AB5320, EMD-Millipore, Biilerica, MA), rat anti-CD45 1:100

( 530539, BD Biosciences, San Jose, California), rat anti-F 80 1 :500 (#MCA497R, Ab^'D Serotec-BioRad, Raleigh, NC), rabbi anti-GFP 1 :400, and chicken anti-GFP .1 :400 (#A1 1 122 and #Ai0262, Life Technologies, Carlsbad, CA). The two GF aatibodies performed, equally well; the chicken anti-GFP antibody was used in the clodronate-liposorae experiment, and the rabbit antibody was used throughout the rest of the study. On da 2, the slides were washed, three limes in PBS, incubated with corresponding 1:400 diluted Alexa Fluor 488- and Alexa Fluor 394-conjugated secondary antibodies ( Jackson Imt ino Research Laboratories, West Grove, PA), and counterstained with SIowFade Gold Ant i fade mounting reagent with 4',6- diamidino-2-phenylmdole (DAPI) (Life Technologies).

itnraunofiupresceace microscopy-based analys is of viral reporter gene express ion immunofluorescence tillages were collected using an Olympus BX61 microscope equipped with an FVil digital camera (Olympus America, Center Valley, PA), The Extended Focal imaging (EFT) function was used in collecting .high-magoiiication micrographs to allow the creation of a single in-focus image from a series of views of the same field at different z- dimeasional focal planes at 2 pm intervals. EFI was carried out in a live-processing mode during image acquisition.

Camera acquisition time for EGFP immunofluorescence was optimized and set a priori for each organ through independent experiments where the collected data were pooled for statistical analyses. The optimized acquisition time for EGFP i.ramu¾ofl orescence display was 200 msec for liver, 400 msec for spleen, 300 msec for lung, 300 msec for heart,

300 msec for kidney, ! sec for muscle. 500 msec for pancreas, 1 see for small bowel, 1 see for large bowel, and 50(1 msec for brain. Wherever a figure contains micrographs collected using a different level of exposure for EGFP, the settin is indicated in the figure legend. Immunofluorescence micrographs were subjected to measurement of both color intensity and color-positive area using MicroSoite Biological Suite image analysis software Version 5 (Olympus). To determine the EGFP fluorescence intensity, a threshold defining the background green fluorescence color for each pixel was set at 70 while the possible range of intensity values were from 0 representing a complete absence of green color intensity to 255 taken to be of full intensity. A region of interest (ROl) was drawn over the tissue

compartment in each image, and positive ID particles in the ROl, defined as containing al least 5 connected pixels w ith above the background color intensity, were identified. The color intensity values from every pixel of positive ID particles were summed and normalized by the tissue ROl area (per pfn2). To evaluate the fraction of tissue vascular area expressing EGFP, the endothelial marker-positive area and EGFP-positsve area within the tissue ROl w re quantified by summing up the areas of positive ID particles based on the color detection threshold of 70 for both the green and red colors. The percentage ratio of EGFP -positive area to EC-positive area in each organ was calculated for evaluating the vascular EC vector gene expression. Mean and standard deviation of data points in each organ derived from experiment mice were plotted.

Quantitat e flow cytometry

Peripheral blood was collected from mice treated with vehicle or clodronafe liposomes and 50 pL of each sample was spiked with re-fluorescent beads (hwitrogen, CA) as internal standard for absolute counts. Red blood cells were lysed with, red blood cell lysis buffer (BioLegend, San Diego, CA), and mononuclear cells (MNCs) were isolated. MNCs were then washed with cold P BS and. stained with CD I lb~f iuoresceta isothtocyanate (FITC) and CD 5-phyeoerythrin (PE) (BD Pharmingen, BD Biosciences, San Jose, CA) for 1 hr on ice. Then, cells were washed, resuspertded in PBS and analyzed by flow cytometry. Forward scatter (FSC) and side scatter (SSC) were used to gate monocytes as high-size (FSC)/ low- granulation (SSC) population; moreover, the monocyte population was further characterized as CD I i b-positive/CD45-positive. The count of the FSC-high/SSC-low/CD! ih- positive/CD45-positive monocyte population was normalized to the count of the -fluorescent beads. Results were presented as the % of average of vehicle treated mice. Statistical analysis

Ail data are reported as mean .-A-, standard deviation. Significance of the means between the mouse groups was determined using unpaired Student's test, for each organ with

Bonier roni correction for multiple independent comparisons carried out on different: organs from the same cohort of mice. Statistical significance was defined as adjusted P < 0.05

(OraphPad Prism, San Diego, CA),

Adenoviral vector construction: Replication incompetent El~ and E3-deleted

Ad5CM V^'-GFP and Ad5Robo4-GFP vectors were created using a two-plasmid rescue method. Embodiment, plasitiids encoded expression cassettes including the human

cytomegalovirus (CM V} major immediate-early promoter/enhancer or the magic roundabout (110604) enhancer/promoter elements coupled to the enhanced green fluorescent protein gene, followed b the bovine growth hormone poSyadenyiation signal. These expression cassettes were cloned into a shuttle plasraid {pShuttie, Qbtogene, Carlsbad, CA) and confirmed using restriction enzyme mapping and partial sequence analysis. The shuttle plasmids were linearized with Pme 1. enzyme and integrated into the AdS genome by homologous recombination with pAdEasy- 1 piasmsd in E. co!i strain BIS i 83. Recombinant viral genomes were transacted into MEK.293 ceils using SuperFect ^"f ransfection Reagent (QIAGEN, Chatsworth, CA), and packaged into virus particles. AdSCMV-GF.P an

AdSR.0804~GFP were propagated in FIEK293 cells, purified twice by CsCl gradient eentrifugation and dialyzed against 10 raM BEPBS, 1 raM MgCh, pH 7.8 with 1 % glycerol. The viral particle (vp) concentration was determined by absorbance of dissociated virus at A260 am using a conversion factor of 1 ,1 x W² vp pe absorbance unit.

Generation of composite mice; The Animal Studies Committee of Washington University in St. Louis approved all procedures. Rag-2 knockout (KG) mice (13), in a mixed genetic background, were bred in-house. Transgenic hCA mice on. a mixed genetic background, likely CS7BI6/J^' and DBA (14), were obtained from Svea Pettersson. ROSA- R26R knock-in mice were obtained in-house. Rag«2KO/ O mice were serially intercrossed with R26R and hCAR transgenic mice to generate the composite mouse line,

hCAR/wi:R26R/R26R;Kag2KO/K.O, termed hCAR:Rag2 O/KO. The R26R conditional LaeZ alleles were not used in these experiments. The warfarin li er detargeting experiments were performed using wt/wtiR26R/R26.R;R.ag2KO/K.O littemtates.

Creation of orthotopic and subcutaneous heterotopic tumors: The 786-0 human kidney cancer cell line was obtained from ATCC and cultured in RPMi with 10% FBS with pen strep/amphotericm B. Xenograft, tumors were established by injection of 5X106 ceils in 50uL of PK51 media usin aseptic technique. Kidney orthotopic tumors were established by left kidney subcapsular injection of 4X 106 786-0 ceils in 40uL of RPMI media, Carprofen, 5mg/kg sc X 3 days, (Pfizer Animal Health, NY, NY) was used for postop analgesia. Mice were injected with Ad vectors when the xenograft tumors reached a diameter of about 4 torn.

Ad vector injections, host organ, and tumor harvest; Mice barboring established subcutaneous and kidney tumors were tail vein injected with 5.0.x 10^U', 1 .0 xl0^H, or i ,5 xlfl" viral particles of Ad5ROB04-GFP or Ad5CMV-GFP in 200 μ.1 of saline. For warfarin experiments, mice were administrated warfarin (5 mg/kg) dissolved in peanut oil subcutaneous!}' on day -3 and day -1 prior to vector injection. Seventy-two hours post vector administration, mice were anesthetized with 2.5% 2, 2, 2-tribromoethano! {Avertin, Sigma- Aldrich, St. Louis, MO) perfused via the left ventricle with phosphate-buffered saline (PBS, pH7.4J, followed by 4% paraforniaidehyde/PBS for whole body fixation. Moose organs and tumors were collected, post-fixed in 4% paraformaldehyde for 2 hours at room temperature, eryopreserved in 30% sucrose tor 16 hours at ^QC, and cryo-emhedded in NBG50 (Thernio Fisher Scientific, Wallham, MA) over 2-methyihutane/liquid nitrogen.

Tissue harvest and inimunofluorescent localization of reporter gene expression: Sixieen-micrometer frozen sections were air-dried, washed in PBS, blocked with protein block ( .1 % donkey serum in PBS containing 0.1 % Triton X- 1 0), and incubated with pri mary antibodies including: rat anti-endomucin, 1 : 1 ,000, (#14-5851-81 eBieseience, San Diego, CA), Armenian hamster anti-CD31 , 1: 1 ,000, {#MABf 3982 EMD-Miilipore, Billeriea, MA), and rabbit anti-GFP, i :400, (MA 1 1 122 Life Technologies, Carlsbad, CA). After PBS washes, the slides were incubated with corresponding Alexa Fluor 488 and A!exa Fluor 594, 1 :400, (Jackson IrnraunoReseareh Laboratories, West Grove, PA) conjugated secondary antibodies and counter/stained for nuclei with SlowFade Gold Antifade mounting reagent with. 4',6- diamidino-2-phenylmdole (DAP!) (Lite Technologies), fluorescence microscope images were collected using an FV.fi digital camera with Extended Focal Imaging (EH) function (Olympus America, Center Valley, PA), To quantiiv the tissue section GFP fluorescence, the areas of GFP(t ) cells and dual CD3 l/endomucin(+) blood vessels were measured and normalized by total tissue area per .field. Areas of positive fluorescence were quantified using image analysis software (MicroSuite Biological Suite Version 5, Olympus).

Tissue and whole organ reporter protein, expression by imsBii.nob1ott.ing: Mice were perfused via the left ventricle with cold phosphate-buffered saline (PBS, pFI 7.4) containing 1 ttiM PMSF (Sigma-Aidrieh). Organ tissues and tumors were snap frozen in liquid nitrogen and stored in the liquid nitrogen vapor phase. Frozen tissues were pulverized using a liquid nitrogen-chilled Cell Crusher (Thermo- isher), and lysed on ice in radioimmunoprecipitation assa buffer (20 raM Tris-HC! (pH 7.6), 0.15 M Nad, 1 % sodium deoxycholate, 1 % NP40, 1 ffiM EDTA, I niM ΕΟ^'ΪΆ) supplemented, with Protease inhibitor Cocktail, 1 : 10, Sigma- AJdrich) for 30 minutes. Protein lysaies were separated on polyacrylamide gels and transferred to poiyvinyhdene difluori.de (PVDF) membranes. Protein loading in individual lanes was normalized first to β-tubu!in and then VE-Cadherin. Membranes were blocked in Tris-buffered saline, TBS, pH 7.6, containing 0.5% Tween 20 (TBST) and 5% nonfat dry milk and incubated in 5% BSA in TBST, containing the following antibodies; rabbit polyclonal anti- OB04 (Dean Li, University of Utah), chicken monoclonal anti-EGFP, 1 1 ,000, (# A 10262 life Technologies), goat anti-VE-Cadherin, 1 :40CS, (# A 1 02 R&D Systems, Minneapolis, MNk and polyclonal anti-f3-tubulin, .1 :2 ,000, (Abeam, Cambridge, MA) overnight.

Membranes were washed three times with TBST and incubated, in BSA 8ST with the corresponding l.gG -horseradish peroxidase conjugate, 1 :5,000, (Santa Cruz

Biotechnology, Santa Cruz, CA) for I hour. After three TBST washes, peroxidase activity was revealed by enhanced chemil¾m.i»escen.ce using EC.1..2 or SuperSignal West Femto Western Blotting Substrate (both from Thermo Scientific) and imaged, using a Chemidoc X^'RS imaging system (Bio-Rad .Laboratories, Hercules, CA). The immunoblotting was quantified by densitometry with Quantity One ones-dimensional analysis software (Bio-Rad Laboratories). Statistical analysis: Significance between groups in the differential fluorescent area experiments was determined using one-way A^' OVA with Takey's correction for multiple group comparisons (Graph Pad Prism, San Diego, CA).

Our molecular and genetic resource tools enable us to obviate hepatotoxicity, innate and adaptive immunity, reticuloendothelial cell (RES) sequestration, and transgene expression persistence. Hepatic sequestration, can be overcome by abrogating Ad eapsid hexon and pen ton blood coagulation factor binding (Waddinglon et al 2008). Warfarin can be used to achieve this in mice in the short term (FIG. 5), but vectors are also available with hexon and pension mutations (Short et ai. 2010). Mutant eapsid vectors are a translational bridge to clinical trials (Kim et a! . 2012). Our strategy of transcriptional. (FIG. 8) or transductional EC targeting circumvents hepatocyte vector transgene expression underlying- liver toxicity (Raper et al. 2003). Further diminutions of innate and adaptive immunity-can be achieved through additional, vector engineering. Our strategy of helper-dependent, "gutless" Ad vectors includes vectors lacking the entire Ad genome save for vector long terminal repeats (Muhammad et al 20.10). The nominal viral D^'NA within these vectors can minimize innate immunity, and the lack of viral, protein expression can evade adaptive immunity. Inhibition of RES sequestration, and pree istent neutralizing antibodies can be achieved by tailored capsid polyethylene glycol (PEG) shielding (Zeng et al. 2012). Gutless vectors can also achieve prolonged iransgene expression (Kim et at 2001 ). Recent clinical trials showed the feasibility of sate, non-toxic Ad vector systemic (I V) administration ( athwatrs et at 201 i ; Brenner et al. 2013).

Examples

The present teachings including descriptions provided in the Examples that are not intended to limit the scope of any claim or aspect Unless specifically resented in the past tense, an example can be a prophetic or an actual example. The following non-limiting examples are provided to further illustrate the present teachings. Those of skill in the art, in. light of the present disclosure, will appreciate that many changes can he made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present teachings.

.Example I

This example illustrates the upreguiation of endogenous ROB04 in renal, cancer xenografts and orthotopic tumors.

To evaluate Ad vectors for tumor EC targeting, a cancer histotype was selected forming hypervaseular tumors both. in. mouse models and in. human disease. Renal cell cancer ( CC) is a paradigm clinical hypervaseular tumor whose principal therapy is drugs targeting angiogencsis. The human, derived 786-0 renal carcinoma cell line was selected because these cells possess the molecular features of, and histologically emulate, clinical renal cell cancer in -patients i ondo K et al 2003; Gordan JD e( «/. 2008). in addition, the cells form hypervaseular xenograft and orthotopic kidney subcapsular tumors (FIG, 3). It was hypothesized that the vascular ECs in 786-0 tumors would be activated (taking into consideration FIG. 7). Warfarin liver deiargeting enhanced the multiplicity of tumor endothelial ceil reporter gene expression in both tumor locales. (FIG. 7) One candidate gene whose promoter element could target. Act vectors for EC specific expression in tumor- activated vessels is ROB04 (Okada Y et at 2007; Ru ninieeki L et aL 2002; Seth P et al. 2005), Up regulation of the R08O4 endogenous gene in RCC tumor models was tested. Extracts were immuoobiotted from 786-0 xenografts, orthotopic tumors, and liver as a. control host organ (F G. 1). The similar levels of vascular endothelial cadherra (VE- Cadherin, Cdh5) expression (FIG. 1 ) combined wit about equivalent vascularity as determined by EC marker immunofluorescence (FIG, 2 and FiG.3) supports the use of liver as a control host organ for comparison, with RCC tumors, Denskwmetric normalization, to VB~ Cadherin revealed a 1.8- fold increase endogenous mouse ROB04 in both xenografts and orthotopic RCC tumors (FIG. 1). in FIG. 2, FIG.3 and subsequent drawings based on multicolor originals, gray- scale versions of each color channel (red, green and blue) are shown, as well as a composite gray scale that combines all 3 ( RGB) color channels. In each case, the top left panel is the red channel, the top right panel is the blue channel, the bottom left panel is the green, channel, and the bottom right channel is the composite.

Example 2

This example illustrates that an Ad5ROB(M vector transcriptionally targets tumor endothelial cells.

To transcriptionally target, an Ad vector to RCC tumor vasculature the 3 kb enhancer promoter fragment of human ROB04 previously validated for endothelial expression in single copy and endogenous locus transgenic knock-in mice (Okada Y ei af. 2007) was used. EC's are known to express trace levels of the Coxsackie and adenovirus receptor (CA R) (Reynolds PN et al. 2000; Preuss MA ei ah 2008). Immunodeficient composite mice were created containing a human CAR (hCAR) transgene and Rag2 gene deletion (Shinkai Y t aL 1 92; Tallone T et al. 2001 ). Reporter gene localization within tu mor ECs was tested (FIG. 3). There was a dichotomy in Ad5ROB04 versus AdSC V vector expression pattern in both, kidney orthotopic (KG) and . subcutaneous (SC) xenograft tumors of mice intravenously injected with 1.5X 101 ! viral particles (vp) (FIG. 2). intense BGFP expression is also detected in endothelial tip cells, hi contrast, AdSCMV -directed expression can be detected in. host, kidney but neither in orthotopic, nor in subcutaneous tumors. Ad5ROB04-direeied expression was restricted to ECs in both kidney and subcutaneous tumors, Ad5 OB04 endothelial reporter expression distribution was reduced in mice injected with lower, 1 X.1^'011 or 5X 10.10 vp, dosages, but EC fluorescence intensity was maintained. There was no detectable eo- localized expression w thin either kidney orthograft or subcutaneous tumors its Ad5CMV-EGFP injected mice despite focal glomerular and interstitial peritubular EC expression in. the adjacent, kidneys of these mice (FiG. 2). AdSROBCM d irected expression was endothelial specific, as neither CD45 cells nor pericytes were positive for EGFP expression (FiG. 2),

Example 3

This example illustrates that an AdSROBCM vector transcriptionally targets metastatic tumor endothelial ceils. Durin tissue immunofluorescence analysis infra-ovarian and peritonea! metastases were detected in an Ad5ROB04 injected mouse (1.5X 10! I vp) bearing an orthotopic tumor (FIG. 3). Nearly all of the microvessels within the intraovarian and peritoneal metastases expressed EGFP. There was almost no expression within stromal ECs within the metastasis- bearing ovary except .for perifollicular microvessels (FIG. 3). Intra-ovarian " rukenberg" renal carcinoma metastases in hCAR:Rag2KO O mice injected with ί .5x 10" vp, FIGS. 3A- 3C, arrowheads, from subcapsular 786-0 orthografts demonstrate extensive and intense rn.ierove_.sseI EGFP expression. Expression was not detectable m EC^'s within the fallopian tube abutting the peritoneal metastasis (FIG. 3).

Example 4

This example illustrates the Ad5ROB04 reporter protein expression in orthotopic and xenograft tumors compared to an index host organ..

To quantitatively test for Ad-mediated tumor reporter expression extracts were immunoblotted from both tumor locales and. liver, from mice injected with 5 10'° vp of either the Ad5ROB04 or AdSCMV vectors, and probed for EGFP normalized to either VE- Cad erin or β-tubulin. Neither the orthotopic nor xenograft extracts contained detectable EGFP protein as evidenced by the validated immtinofluorescem^' absence of detectable tumor AdSCMV regulated expression (FIG. 2). AdSROBCM-mediated EGFP expression was 2-2,4- fold elevated when normalized, to β-tubulin and.2.6-2.8-fold elevated when, normalized to VE-Cadherin (FIG, 2). AdCMV-directed liver expression was 7- to nearly ί 0-fold elevated when normalised to ji-tubulin or VE-Cadherin respectively, compared, to Ad5 .O 04- regulated expression. (FIG. 2). This result demonstrates the ability of EC transcriptional regulation to detarget Ad hepatic expression.

Example 5

This example illustrates endothelial specific reporter gene expression mediated by both Ad5R()B04 and AdSCMV vectors after systemic injection in hCAR transgenic mice,

Ad5ROB04 mediated vector expression was tested using immunofluorescence in a nine organ pane ! of ^'host organs from the same tumor bearing h.CAR:Rag2KO KO mice as in FIG . 2. Endothelial expression was detected in long, kidney , muscle, adrenal , heart, skin (FIG. 3), and brain (data not shown) of mice injected with either vector. Both liver and spleen displayed differential cell ty pe localized, reporter gene expression mediated by Ad5ROB04 versus AdSCMV vectors. In liver, Ad5ROB04- directed EGFP expression, was confined to sinusoidal ECs, whereas AdSCMV -directed EGFP expression, was focal ly detected, in hepatoeytes. In spleen, Ad5RGB04-directed expression was also EC restricted whereas AdSCMV-directed expression was localized to marginal, x ne CD1:6/CD32 F4 80( .| retkuloendothehal cells (FIG. 3), The dose dependency was examined of both the

Ad5 OB0 -, and Ad5CMV-EGFP vectors due to EC expression of AdSCM^'V-EGFP (in some cases adrenal heart . muscle). Injection of 5X1010 vp of either vector into

h \R;Rag2KO/ O mice demonstrated a reduction of heart, kidney, and brain, endolheiial expression mediated by either vector, and a decrease w ith retention of adrenal endothelial expression with either vector (FIG. 4). Host organ EGFP reporter expression following intravenous injection of either Ad 5 OB 04 - EGFP (ROB04) or Ad5CMV-EGFP (CMV). Fig, 4 illustrates injection of 1.5 l0^u viral particles (vp> produced extensive microvessel EGFP expression in both Ad5R0804 and AdSCMV vector treated mice in kidney, lung, muscle, adrenal, heart, and skin. In liver and spleen AdSC V -directed EGFP expression was localized to reticuloendothelial system (RES) cell in. con rast to micro vessel restricted Ad5ROB04 directed expression. Lung, liver, spleen and muscle maintained vector specific expression levels and patterns seen with the higher vp dose (FIG. 4).

Example 6

This example illustrates that liver detargeting in ag2f O mice abrogates promiscuous host organ EC Ad5R.OB04 reporter expression.

The ability to inhibit liver viral particle sequestration by warfarin-mediated blood coagulation factor depletion was used in hCAR(-) wild type mice (Waddmgtori SN et al 2008; Alba R ef. al 2010) to demonstrate target cell vector payloa expression in the context of low hCAR expressing F.Cs. Liver detargeiing efficiency was tested in our Ragi O/ O mice. Warfarin pretreatmeut on day -3 and -1 before injection of IX !0^U vp AdCMV-EGFP, revealed a diminution of hepatocyte reporter expression (FIG. 4).. It was tested whether warfarin pretreatment in the absence of the hCAR transgene would produce host organ AdSR.0.804 EC expression (FIG. 5) in RCC tumor-bearing mice. Compared to the hCAR mice, warfarin treatment either failed or barely produced detectable tissue reporter immunofluorescence in seven of nine host organs (FiG. 5 A and data not shown (brain)). Similar to hCAR transgenic mice, there was focal, scattered liver and splenic EC expression in warfarin-treated, Ad5R0804 injected, B.ag2KO/KO mice (FIG. SA).

The discordance between imniunoftuorescence localization and intensity between host organs of hCAR transgenic compared to warfarin treated Rag2 O/KO mice, motivated a more quantitative analysis of Ad5ROB04- ediaied reporter gene expression. Host organ i mmu nohlois revealed negl i gible expression in five of seven organs eit her with or without warfarin pretreaiment. Warfarin produced a p nounced in version of splenic versus hepatic reporter gene expression (FIG, 5B).

Example 7

This example illustrates that AdS OBCM EC targeting is maintained and

differentially enhanced in both orthotopic and xenograft tumors compared to host organs following warfarin-liver detargeting.

As liver detargeting effectively eliminated host, organ Ad5RO804 EC expression, our next question was whether tumor EC expression would be similarly diminished. Consistent with baseline endogenous ROB04 protein expression (FIG. I) scattered EC reporter expression was detectable in both orthograft and xenograft RCC tumors even in vehicle- treated mice (FIG. 6). Warfarin pretreatmem produced increased EC reporter ex ression at both tumor locales. Warfarin pretreaiment increases splenic EGFP EC expression however ail other organs except liver display sporadic or no reporter immunofluorescence. (FIG. 6} To quantify frequencies of immunofluorescent EC reporter gene expression, in tumor versus host organs in the presence or absence of warfarin image analysis of tumor sections from 3-4 mice were used (FIG. 5). Warfarin produced a eight-fold increase the EGFP(-f ) compared to EC marker area in orthografts and a six-fold increase in xenografts (FIG. 5). Fi ve of seven host organs evidenced minima! expression, albeit with single outlier mice in each organ. Warfarin produced a 1.7-fold decrease in hepatic and a 2.6-fofd increase in splenic expression.

Iramunoblotting of liver extracts ifom Ad5 OB0 injected mice (FIG. 6C) revealed a fourfold decrease of liver (EC localized, FIG , 6A) EGFP expression normalized to tubulin and a two-fold expression decrease normalized to VECadberin, m contrast tumor EGFP protein expression increased 1 ,4-fold at both sites following warfarin pretreaiment. The splenic expression is markedly increased, by warfarin whereas liver expression is decreased, (FIG, 6) Collectively, these data demonstrate the tumor EC selectivity of the Ad5ROB04 vector. Ex am le 8

This example illustrates Ad. vector expression in ECs, generating active drug with secretion into the bone marrow,

in these experiments, an EC-specific vector configuration contained 3 kb of the human Magic Roundabout (ROB04) enhancer promoter. ROB04 is specifically expressed in ECs. it was confirmed that the EC specificity using an Ad.SROB04-EGFP vector. This vector was expressed in tumor neovascu!ar ECs, liver, spleen, and bone marrow sinusoidal ECs.

Another vector configuration included Ad5ROB04-EGFP with a bacterial cytosine deaminase prodrug converting enzyme that can produce the cytotoxic ehemotherapeutic, 5- tluorouraeil (5.FU) from S^luoroeytosine (S-FC). EC-generated 5-FU ablated host bone marrow hematopoietic cells. The Ad vector configuration was exclusivel expressed In ECs, generating activ drug with secretion, into the bone marrow mieroenvironme t to achieve host ceil killing.

Example 9

This example illustrates mobilization of granulocytes, monocytes, and lymphocytes from the bone marrow to the peripheral circulation and the spleen, with a

Ad5ROB04sCXCR42-28 vector.

In these experiments, an AdROS04 vector configuration containing a transgene encoding a truncated CXCR4 receptor (an example of a "decoy receptor") was constructed to affect angiocrine adjacent tissue modulation. This ehemofcrae receptor exclusively binds and is acti vated by the cherookine stromal derived factor- i (SDFl). The truncated transgene encodes an SDFl "Hgatxi trap" that is engineered io sequester SDF I from CXCI14 expressing cells, intravenous injection of this d5ROBQ4sCXCR42-2^'8 vector produced mobilization of granulocytes, monocytes, and lymphocytes from the bone marrow to the peripheral circulation and the spleen. These data are consistent: with EC angiocrine secretion of^' sCXCR.4 in the hone marrow, and breaking the attachment of CXCR4 hematopoietic progenitor ceils from their CXCR4 mediated one marrow niches.

Example 10

This example illustrates selective targeting of ECs with an MBO-Ad5 vector configuration.

In these experiments, an Ad vector was created that can selectively target; ECs via vector transduction. This vector was based on our discovery of "myeloid binding protein'^' (MBP) on the surface of myeloid, cells that avidly bound to Ad vectors expressing phage peptide libraries inserted on the Ad vector fiber-knob. An. Ad vector was created that was "deknobbed," and contained a chimeric AdS-T4 phage fibritin shaft and triroematioo domain displaying the MBP peptide, in contrast to the MBP myeloid, binding, the MBP-Ad.5 vector selectively transduced ECs. Results included EC specific MBP~Ad5-EGFP expression in multiple host organ ECs including expression in brain EC^'s and. expression within kidney ECs. The brain and kidney EC targeting have tremendous therapeutic implications for glioblastoma, Alzheimer's, multiple sclerosis, A L.S, and for glomerulosclerosis, .and interstitial renal nephritis.

Ex m le i I. This example illustrates the ability of MBP~Ad5 vectors to target specific tumor microenvironments using tumor-specific tuned promoters.

In these experiments, an Ad vector included tumor EC targeting with this MBP vector using the ROBCM enhancer/promoter fragment. The EC specificity of the MBP-Ad vector was conferred via vector entry (transduction). Transgenes can act as "payloads" into the MBP-Ad vector, which con tains DN A enhancer/promoter elements that are "tuned" to the tumor nncroenvironmeni MBP-Ad vector configurations including 'tumor tuned" promoters can transduce multiple host and tumor EC^'s, but solely expressed in tumors due to characteristics conveyed on their associated and embedded ECs. These tumor EC specific characteristics can include but are not limited to activation by hypoxia, D A damage stress, endoplasmic reticulum/unfolded protein response stress, and redox/free radical stress. EC angiocrine engineering can tailor solely to the tumor niieroenvironnieni to enhance potency and specificity by arming MBP--Ad5 vectors with tumor-specific tuned promoters.

Example 12

This example illustrates testing for PCA bone metastases growth inhibition due to dysregulation of CSC bone niche cellular components by angiocrine targeted prodrug- converting enzyme expressing Ad vector configurations.

Host sinusoidal capillaries are principally composed: of ECs. therefore the BM niche components can be particularly .susceptible to angiocrine targeted Ad vector configurations. One example of the EC-niche cell spatial relationship is the localization of the principal SDF1 (CXC1..12) producing BM niche component the C.XCI..12 Abundant Reticular (CAR) cell (Omatsu ei al 2010; Greenbaum ei ni, 2013). In these experiments, immunofluorescence was used to determine the EC-CAR spatial organization, in the femur. The data demonstrate the investment of bone sinusoidal vascular ECs by CAR-EGFP cells (FIG. 12). Angiocrine- prodtreed 5-FU (FIG. 1.1 FIG. 14, FIG, IS) can dysregis!ate the host bone marrow niche to effect PCA CSC depletion via niche eviction and quiescence abrogation. FIG. 13 illustrates an embodiment of an EC targeted prodrug-converting enzy me Ad vector Acl5R0BO4- bCDD3 l 4A. The bacterial eytosine deaminase (bCD) cD A contains an aspartate-alanine substitution (D314A) enhancing 5-fiuo.rocytosine (5-FC) to 5-fiuorouracil (5-FU) conversion. The principal R A processing dysregitSation mediated by 5-FU can enable functional disruption of quiescent bone niche components. There was an A.d vector expression gradient between intra and perif umorai ECs and distal uninvolved bone marrow (FIG. 10), that supports differential CSC versus HSPC bone niche targeting. Focal intratumoral EC production of the stem eel! Ligand decoys can permit selective mobilization of PC A CSCs compared to host HSPCs. A collection of lineage-restricted transgenic reporter mice can be used to elucidate the distinct niche cell type targeted by angiocrine 5-FU production. Physical relationships, between lineage-marked cells and metastatic PCA ceils can be established and preferential sensitivities of niche cellular components and host H^'SPCs that are spatially dependent or independent of angiocrine 5-FU production can be tested. Differentia! PCA and niche lineage ceil fluorophore marking can be used for frequency enumeration, quiescence, proliferation, arid apoptosis analyses. Cell sorting can be used for candidate gene and unbiased expression profiling focusing on secreted and membrane-tethered molecules directing CSC-niche maintenance potentially dysregulaied by angiocrine 5-FU production. Engineered PCA cells that can report on quiescence versus proliferation allow for the determination of the disruption, extent of angiocrine 5-FU on niche CSC maintenance. Deployment of ECniche cell culture modeling (Seandel et ah 2008: Butler et id. 201 ; obayashi et at. 2010) can allow further delineation of the mechanisms of angiocrine-CSC disruption.

Bone sinusoidal ECs can be exploited to produce and then secrete our prodrug product, 5-FU into the bone niche .microenviroameni. Focal 5-FU can differentially dysregulate host cellula niche component^'s embedded within PCA metastases compared to uninvo!ved bone .marrow regions. EC specificity and tumor bias was validated of the Ad5ROB04 vector (FlG.s 8- 10), and. target vector Ad5ROB04-bCDD314 A embodiment was created (FIG. 13). bCDD314A is a bacterial derived cytosine deaminase containing an aspartate to alanine point mutation. bCDD3 !4A possesses a marked increase in 5-FC-5FU conversion activit compared to wild type bacterial or yeast CD (Fnchiia et l 2009) CDuarte et a I. 2012). An experiment with Ad5-bCDD314A IV injection in Rag2KO miee bearing 786-0 RCC xenografts was performed. bCDD314 A transgene activity of an AdSC V vector that is expressed in liver and spleen was tested. Despite warfarin-mediated liver de-targeting (FIG. 5),

Ad5CM '^"bCDD314A treated mice lost weight after 4 days of twice daily 5-FC, 500 mg kg ip (FIG, 1 ). . An embodiment Ad5ROB04 vector doubled the 5-FC tolerance. Further dose reduction eliminated phenotypic toxicity . Warfarin on day -3/-1. and Ad vector injection on. day 0. (Fl^'G. 14) The induction of toxicity in the mice was in striking contrast to studies wherein IV AdCMV-bCD injection decreased hepatic colonic metastases growth while sparing host hepatocyte function (Topf et l. 1998). This discrepancy can be due to an enhanced potency of the bCDD3 l 4A transgene compared to the wild type counterpar. In these experiments, warfarin pretreaied .mice injected with Ad5ROB04-bCD.D314A appeared unaffected until weight loss starting on day 9 of 5-FC treatment (FIG. 14). At sacrifice on day ί I, examination of the bone marrow revealed focal ablation of hematopoietic elements in the Ad5ROB04-bCD injected mice (FIG. 15). At sacrifice on day 1 L examination of the bone marrow revealed local ablation of hematopoietic elements in the Ad5ROB04-bCD injected mice. (FIG. 15) Analysis of RCC xenografts revealed apoptosis and necrosis in the vector-treated compared to untreated tumors. A dose redaction test as performed for a nontoxic 5-FC dose in n ntumor bearing Rag2KO rake (F G. 14). The results demonstrate that angioerine hCD 5-FU production can be accomplished without host toxicity. These results support the in vivo functionality of the bC.DD31 A transgene, and that angioerine targeted Ad vectors can affect the bone marrow mieroen ironraent.

Example 13

This example illustrates, testing of angiocrme-iargeted prodrug dysregulatmn of bone marrow niche supporting ceil lineages.

fn these experiments, a prioritized panel of lineage marked once were interrogated (FIG. 1.6). Prioritization can be based on distance from bone marrow sinusoidal EC^'s. CAR cell frequencies and perivascular locale alterations can be tested and quantified, tor anatomic and morphological localization within .metastatic tumors and unin vol.ved bone marrow using tissue section, immunofluorescence image analysis and flow cytometry gated on GPP, CXCR4, and VCA cell surface markers (Qmatsu et ai, 2010), CAR ceil functional alteration can be tested by bone marrow SD.F1 ELISA. CAR cells are the predominant SDF.i source (Oraatsu et ai 2010) but other bone niche components, such as osteoblasts (OBs), mesenchymal and endothelial cells can additionally contribute to marrow SBF 1 production {Greenbaum et ai 2013). To further test for 5-FU mediated CAR cell functional impairment, GPP flow sorted CAR cells can be cultured in adi oogenic or mesenchymal media, the former to test adipocyte differentiation and the latter testing for colony-forming cel!-fihroblast (CFC- F) generation (Omatsu et ai 2010; Greenbaum et at 2013), These assays can provide mechanistic insight into how angiocrine-targeted 5-FU production alters CA ceil function. As nestin(t ) cells also abut bone sinusoidal capillaries they can be used for lineage tracing (FIG. 16) (Nagasawa el ai. 201 1). Prxl is a marker of mesenchymal _.progenitors/stem ceils CMSCs) (Logan et ai. 2002). Prxl cells are also requisite niche components (Ding and

Morrison 2013; Greenbaum et ai 2013).. Moreover, recent data suggest that MSCs contribute to bone metastatic progression in general (Koh and Kang 2 12), and are an additional source of SDF 1 CXCL12 production in particular (Ye . et ai 2012; Borghese et ai 2013; Mognetii et αί 20 ! 3). Osteoblasts (OBs) have also been suggested as crucial PCA/CSC niche

components (Chung et al 2009; Schulze el al 2010; Fritz <?/ « . 201 1 ; Sehulze et al 2012). Angiocrine-S-FU effects on OBs will be determined using Col2.3- OFF lineage tracing (FIG. 16). While OBs are not as mtimateiy associated with ECs as CAR cells, a sinusoidal capillary- subset closely approximates the OB-enriched bone surface and as such, could be impact this endosteal niche. Flow-sorted Pr i/ SC and Col2,3/OB GFPf ) cells from 5-FC treated mice can be tested tor differentiation and bone formation perturbation in cell culture assays (Weilbaecher and Novack LOSs) as described above. for CAR cells (Su et al 2012; Yang et ai. 2013),

A corollary to angiocrine 5-FU niche dysregulation is perturbation of PCA CSC maintenance, abrogating CSC quiescence eventuating in CSC depletion and prolifer ti e transit amplifying cell population expansion. Multiparameter immunofluorescence can be engaged using PCA CSC and HSC stem and differentiation markers in both tissue sections and flow cytometry. Approaches to functionally report on. CSC and. HSC quiescence and proliferation can also be used. Stem cell quiescence detection data can be used from on bromodeoxyu ridi ne label retention.

Dilution of a chromatin-bmding histone 2B-GFP (H2B-GFP) fusion protein can be used to estimate CSC quiescence (Kanda et al. 1 98; Hadjantonakis and Papaioannou 2004; Wilson i'i ai. 2008). Labeling can be performed using different 02B-tluorop.bore colors to assay both populations in the ame mouse (Hadjantonakis et al. 2003) (FIG, 17). A lentiviral dual .r lA/T E "tight" TetON-histone 28 (H2B)-mChe.rry virus can be constructed like the TetOff system (Palkowska-Hansen et aL 2010). IGR-CaPl cells can be ientiviraily infected with TetON-H28-mCherry and CMV-pLUC and select DOX induced reporters and

constitutive LUC expression,

TetOP-lilB-GFP mice bitransgenic can be obtained for both the tlTA TetON operator and TREH2B-GFP iransgenes (Foudi et. al 2009} (JAX) and intercross with Rag2KO mice. DOX-pre-induced iGRCaPI : TetO -H2B-mCherry cells can be intracardiac injected into DOX prefreated Te tO -H 2 B -G FP : Rag2 KG mice (FIG, 17). The six-eight week lag time for IGK-CaPi gross bone metastases development can allow for a DOX withdrawal washout period to test for H2B label retention consistent with stem and early progenitor cells. PCA CSC versus HSC quiescence can be quan ified by tissue and flow cytometric enumeration of red (CSC) and green (HSPC) fluorescence. Additional testing for differential BSC mobilization and repopulation capability can be performed. Conventional bone tissue section and flow cytometric immunofluorescence can be used to interrogate changes in the metastatic tumor and the bone marrow niche cellular composition, Tissue PCA versus host cellii!ar areas can be tested for proliferation, cell death, and EC vascular marker-imrounofluorescence. To facilitate CA bone localization, add to proiiferatiors apopiosis and flow cytometry enumerations a constitutive KTR-CaPl CMV- H2B-mCherry:LUC (Addgene) cell line can be created. Alterations in PCA CSC versus PCA progenitor or more differentiated PCA cells ca be determined by CD 133, CD44, EpCAM, CP49E CK.5 aod C 8 immunofluorescence co-localized with iOR-CaPl :H2B-mCherry expression. PCA hierarchical composition can be more precisely quantified by flow

cytometry. Dissociated bone tumors can be gated on mCherry, the epithelial identity of those gated cells confirmed by EpCA , then sublVactionated based on CK.5 (basal) versus C 8 luminal, then further fractionated based on CD44 and CD49f,

EGFI^>/:EpCAM:CD44highCD49fC 5h!gh:CD81ow can be presumed to be stem cells. HSC/H^'PC frequencies can be screened using the JKLS C 150+/CD4ii-/FLK- pane! (Mayie e at. 2013). The inverse marker distribution can be designated luminal cells. To investigate whether our EC targeted Ad vector is truly affecting an angiocrine rather than a systemic henotype, .metastatic tumor, BL^'l-iden ified uninvolved bone marrow, blood, and multiple host organs can be tested for 5~FU levels by tlPLC (Kievit et aL 2000).

Example 14

This example illustrates cell culture experiments to test PCA stem cell potential PCA stem cell potential can be functionally tested. 5-PU can decrease the frequenc of mtratumorai CSCs and can impair CSC renewal function. Prosiaspheres are considered one hallmark of PCA CSC capacity (Azuma ei. <tl 2005; Guo et aL 2012). Prostasphere renewal capacity can be tested using serial culture. Ability to generate proliferative progeny can be tested by scoring prostasphere size attainment. CSC renewal capacity can be tested using serial limited dilution and serial transplantation experiments (Qin ei al 2012). In addition to these stem cell potential assays, solo and co-culture experiments can also be engaged testing for S-FU-mediated changes in EC-tomor or EC-tumor-osteoblast cross talk. To create an. EC cell culture niche, the Ad5E4~OMPi transfected BUVEC model can be used (Seaodel et al. 2008). These E4-QR El HUVECs ean be maintained for multiple passages, and support HSCs for prolonged eel! culture periods ( obayashJ e/ at 2010). To test for angiocrine functional alterations, 5-FC treated E4-O.RF1 transfected HUVECs infected with AdROB04-bCD or control vectors can be interrogated for differential growth factor and ehenio/cytokine secretion using commercial proteoroie antibody arrays. Array data will be validated by Western blotting and BLISAs. Tumor-EC co-cultures canbe established by '"parachuting'' IGR-CaPi tumor ceils onto E4-ORF-EC cord lattices. Tumor and BCs can be preSaheled with different fluorescent dyes and global gene expression and proteomic secretion alterations profiled from FACS sorted populations.

Data can be produced from at least 4-6 mice injected with experimental, and 4-6 mice injected with control Ad vectors for statistical analysis. Tissue cellular EGFP expression frequencies can determined by measuring the F.C-colocaiized EGFP positi ve area compared to total section area. These area ratios can be obtained from the average of 4 sections per mouse. Celt culture experiments can be repeated 4-6 times as can limit dilution tumor formation analysis. Statistical significance testing can use the non- arametric Mann- Whitney li test, and one-way A OVA.

Example 15

^'This example illustrates testing for PCA CSC versus host HSPC mobilisation, niche depletion, and cytotoxic chemotherapy enhancement mediated by angiocrine targeted Ad vectors expressing stem cell ligand decoys.

PCA CSCs can be regulated by several, stem cell receptof ligand signaling modules, including CXCR4/CXCL12 (SDFI ) iSmi ei al. 2005; Shiozawa i at. 20! ί : Dubrovsk - ei ah 2012), NOTCH/Jagged Delta (Leong and Gao 2008; Wang el ai 2010; Ye at al .201.2), and

W T/Prizzled (Horvath el at 2007; Schweker ei at 2008; awano et ai 2009; Takebe el ai 201 1 ), The CXCR4- SDFI axis can be targeted. CXCR4-SDF S decoy data can be used as a template tor testing of angiocrine Ad vectors slated for NOTCH or WNT ligand decoy signaling disruption. Our data revealed peritumoraJ. EC Ad vector expression with a . gradient diminution in distal metastasis-bearing bone (FIG. 10). intra- and peri -tumoral EC ROB04 promoter acti vation can produce focused CSC mobilization while preserving retention of host HSPCs in nninvolved bone regions. There is extensive expression of the .EC-targeted Ad vector within and adjacent to !GR-CaP I bone metastases. The Ad vector expression gradient between intra and periftimorat ECs and distal unmvolved bone marrow is support for differential CSC bone niche targeting, intra- and peri -tumoral EC ROB04 promoter activation could produce focused CSC mobilization while preserving retention of host HSPCs in uninvolved bone regions (FIG . 10).

A soluble, truncated "sCXCfM" expressing Ad vector was created. A AdSCM V-sCXCR4-Fc was constructed and activity tested (see FIG. 14 for ROB04 vector). The vector iransgene encodes amino acids 2-28 of human CXC.R, which is the SD.F1 ligand binding domain, fused to a mouse immunoglobm. heavy chain (Fc) fragment The vector was validated for mammalian cell expression following virus infection in cell culture and in the plasma of tail, vein injected mice. Systemic C V-sCXC 4 vector injection inhibited B167F10 mouse melanoma lung metastatic implantation and growth {FIG. 18). Systemic C V-sCXC 4 vector injection, inhibited 816/FI 0 mouse melanoma lung metastatic implantation and growth. (FIG. 18). The EC targeted Ad5ROB04-sCXCR4-Fc vector embodiment (FIG. 19} was created and tested. The insert contains the cD A encoding the CXCR4~SD.F1 iigand- binding domain linked to mouse immunoglobin heavy chain for secretion and stabilization , (FIG. 19)

in these experiments, warfarin-pretreated, aontumor bearing, CS7BI6 ^j and Rag2K0 mice were IV injected with Ad5ROB04-sCXCR4-Fc and a. control Ad5ROB04-BGFF vector. Flow cytometry demonstrated elevations of granulocytes, monocytes, and lymphocytes (likely bone resident B-ly.raphocytes) in blood (B) and spleen (S) of Ad5ROB04-sCXCR4 injected mice (FIG, 20). Our results showed expression of A45RQB04 in bone marrow ECs. The results suggest^" Ad vector hijacking of EC ahgioerine functions mobilize ceils from the bone marrow. The BM Ad vector expression gradient (FIG. 10} can focus and differentially amplify CXCL12 sequestration intra and per imetastatieally. Our l X! 0ⁿ viral particle dose has the d namic range enabling ample decremental dose titration, to achieve selective CSC mobilization.

Example 16

This example illustrated testing angiocrine-targeted stem cell ligand sequestration mediated dysregulation of CSC bone marrow niche retention.

Testing can he performed to determine that angioeri.ne stem cell ligand sequestration can differentially mobilize and deplete CSCs vs SPCs, that the angioerine-mediated CSC mobilization can affect loss of CSC compared to HSC giiieseertee and that angiocrine- mediated CSC mobilization can enhance sensitivity of PC A metastatic growth to dpcetaxel.

To test for CSC mobilization, mice with BLI-venfied PCA bone metastases can be l^'V-mjected with Ad5ROB04-sCXCR4, Blood can be analyzed b human Aln RT-PCR (Shtozaw el al 2011 }. If positive, blood PCA ceils can be further enumerated by historic 2B(H2B)-mCherry flow cytometry (Shiozawa e( at. 201 .1 ; Qin f aL^' 2012). IfmCherry labeled cells are detected at sufficient frequency in whole blood, further enumeration of CSCs using our battery of stem ceil, markers can be performed. Companion bone marrow (BM) analyses can test for PCA CSC diminution by flow cytometry ofH2B-mCherry-gated single cell suspensions of bone metastases additionally stained for PCA CSC stem cell markers. Potential shifting, of metastatic PCA quiescence to enhanced proliferation can be initially determined by Ki67 flow cytometry. "Unmvolved" bones suggested by Bl.i can also be tested for FCA CSC multiplicity and quiescence/proliferation shifting by CSC stem marker and Κίό? whole BM analyses. Blood. (PC A marker) and bone marrow (PCA-CSC markers) markers can be used as enumerations as benchmarks for decremental vector dose titrations if necessary to achieve differential CSC versus HSPC- niche mobilization. To test for vector host BM mobilization elevations of hematopoietic elements in blood and spleen can be tested. To pinpoint HSPC versus differentiated cell mobilization, colony forming unit-cell (CFC-C) potential in whole blood can be tested. Further HSPC analyses can enumerate BM FISCs and MFCs using SLAM markers (Kiel ef. ah 2005; M^'ayle el at 2013), line HSC population can be functionally characterized for Ad-sCXCR4 vector mediated long-term versus short term, LT- HSC and ST-HS frequencies in mouse reconstitotion assays (Oreenbaura el at 2013;

Mayie et ah 2013), These can be benchmark data from which CSC vs HSPC decremental vector dose titration is evaluated. Flow cytometric analyses of CSC vs HSPC BM

mobilization and depletion can be liniher analyzed using multiparameter tissue

immunofluorescence fo proliferation markers, BrdU, Ki67, and a panel of CSC and HSPC markers. Differentiation of PC A versos host bone marrow elements can be facilitated by H2B immunofluorescence that produces a intense signal with low background. (Hadjamonakis and Papaioaunou 2004).

To test for loss of CSC quiescence mediated by our Ad vector stem ceil ligand.

decoys, the dual color H2B washout experimental strategy can be engaged, as detailed in (FIG. 17). DOX-preireated PCA cells containing the Tetf)N-H2B-mCherry-LlJC virus can be injected into DOX-pretreaied TetO.P-H2B~CiFP:Rag2 O recipients (Fondi e( at 2009;

Falkowska-Bansen el al 2010). Differential retention of BM niche green red fluorescence following 6-8 wk washout can be enumerated by flow cytometry and tissue

immunofluorescence, bolstered by additional markers (Foudi et al 2009). Tumor progressio can be tested using BLl in a group of mice following vector injection, and extended duration experiments testing for overall survival. T¾e question can be addressed of differential CSC specific mobilization mediated by the $CXC 4 vector compared with the "gold standard" CXCR4 small molecule inhibitor, AM.D3.i00. Each vector experiment can include an AMD3100 Ateet ump control emulating continuous vector- mediated sCXCR4 production.

Ad5R.OB04 vectors can be constructed and cell culture validated containing soluble NOTCH and W T ligand decoys (s OTCH and soluble Frizzle Related Protein (sFRP) receptors (Funahasbi et at. 2008; Lavergne et «/. 201 1 ). The existence of NOTCH and WNT pathway c-DN and transgenic reporter mice can expedite efficacy screening and if necessary decreraental Ad vector dose titration both in ceil culture and in intact mice. Additional experiments can use the sCXC 4 experimental template, testing the degree of differential CSC versus host stem cell mobs f ixa i , CSC depletion, and potential PCA tumor growth inhibition achieved with the sNOTCH and sFRP vectors. With the sCXCR.4, sNOTCH, and sFRP data the vector can be selected with greatest CSC functional efficacy to carry forward for additivity testing with cytotoxic chemotherapy. Following the leukemia paradigm ( ervi et αί 2009; Essers and Trumpp 2010), sequential Ad-sCXCR4 or control Ad-LUC vector can be combined with "standard of care" docetaxel chemotherapy (Seruga and Tannock 201 Ϊ ). Additional data is available tor iGR-CaPl cell docetaxel sensitivity... Docetaxel can be given for 2-4 weeks after .Ad vector injection. Tumor growth inhibition or regression can be followed by BLL Blood can be serially sampled for PCA, C'S, and HSPC frequencies as detailed for Aci-sCXCR4, BM and spleen can be analyzed for PCA CSC, HSPC frequencies using flow cytometry and tissue histopathology; proliferation, apoptosis, and vascularity can be tested using multi-marker tissue immunofluorescence bolstered by Western blotting.

To further test for Ad-stem cell ligand decoy CSC niche eviction and consequent depletion cell culture experiments can b used. CSC abundance ca be interrogated by the conipara.ti.ve quantity of prostaspheres formed from. Ad-stem Cell ligand decoy versus control, vector injected mice. CSC renewal capacity can be tested by serial culture. Limited dilation single and serial tumor transplantation experiments can further investigate CSC numbers and functional capacity (Qin et al 2012),

The angiocrine-targeted Ad vector strategy can be differentially localized in metastatic rather than uninvolved bone {FIG. 10), Thus, the Ad vector embodiments can focus CXCR4 blockade to tumor specific, rather than global bone marrow niches. Focal Ad vector- mediated sCXC 4 expression can selectively or preferentially affect CSCs rather than, host HSCs HPCs. The Ad vector system is tunable in regards to promoter selection. Dose titration, or vector sw tching to our EC tropic MBP vector that can contain

enhancer/promoters with greater tumor microenvironment. responsiveness, can achieve a specificity level exceeding global small molecule therapies..

The angiocrine Ad vector approach is also po!y-hgand targeting. This targeting is relevant to CSC-niche crosstalk, as multiple ligand/reeeptor modules can to control PCA CSC maintenance ( arhadkar et al 2004; Chang ei al. 201 1 ; Valdez et al 2012; Ye el al 2012), I ^'XI ligand decoy combinations, or decoy collections collectively as single vector polycistronie combinations can be tested. Switchable promoter elements can be introduced within high capacit " utless^** vectors. The "iheranosiic" attractions of gutless vectors can be further tested.

Example 17

This example illustrates testing theranostic poiyctstronic "gutless" Ad vectors for bone metastatic therapeutic and imaging efficacies.

Polycisironic vectors are emerging as enticing tools for regulation of complex biological processes. Premature nascent peptide release from the ribosome mediated by viral 2A peptide sequences allows for 1 ; i expression of tandem cDNAs (Szymczak-Workman et el. 2012), There are 2A peptide sequences from several viral species that are used in polycisironic vectors. Rules for their sequence ordering within the vector have been established (Sxymezafc- Workman et at. 20.12).

Previous work has demonstrated that polycisironic vectors can rescue quadra-T-cell receptor s bimit knockout mice (Szymezak ei al 2004) and reprogram iPS cells (Carey et al 2009; Shao et l. 2009). Polycistronic vectors have been used in first generation Ad vectors, and can be used for high capacity '' utle s" vectors with their 37 kb capacity (Stadtfekt et al. 2008 ). Switchab!e control of gene products can be implemented. S ilchable control can be applicable to SDF1 -CXCR4 blockade wherein prolonged blockade produced, paradoxical bone metastatic tumor growth en ancement due to ost∞c histogenesis stimulation (Hirbe ei al 2007). Gutless vectors can achieve iheranostic agent swilchable control to allow for cyclical therapeutics when disease recurrence is vector detected. Single, 1X1 vector combinations of our ligartd decoys can be engaged. Combinatorial transgenic mouse and mfeetable/lransfectable NOTCH and WNT reporters for Ad vector-mediated pathway signaling downregu!ation. can be engaged {FIG. 21). Combinatorial transgenic mouse and ittfectabie/transfectabSe NOTCH and WNT reporters for Ad vector-mediated pathway signaling downregulaiion. (FIG. 2.1 ) The CBF 12B-Venus and the TC /LEF-H2B-GFP constructs report on NOTCH and WNT respectively (Ferrer- Vaquer et a 201.0; Nowotschm et al. 201 3). The cDNAs used for transgenic mouse construction are available from Addgene. IGR-CaPi cells can be infected with these constructs and used, for generation of bone metastases. Flow cytometric analyses of reporter fluorescence intensity can be used to to determine the efficacy of Ad vector mediated NOTCH or WNT pathwa downregulaiion (FIG. 2 ! )... To test for comparative Ad vector-mediated host pathway downreguktion each transgenic mouse reporter (J AX available) can be obtained and multiparameter BM. flow cytometry costained with SLAM and lineage markers can be used. PofycisUouic vector configurations can be eonstracted and tested. An embodiment of this vector is presented herein (FIG. 22). Design features of tins vector can include but are not limited to pofycistron EC -targeting via the ROB 4 enhancer promoter, constitutive expression of LUC for BLl bone metastases growth, inhibition, or recurrence detection and EGFP .for enhanced tissue immunofluorescence localization, constitutive prodrug converting, enzyme expression that is functionally conditional due to prodrug dependence, and/or switchable doxycyeline control of multiple stem ceil ligand decoys (Xiong et xL 2006). Design features of thi vector embodiment can include: I ) pofycistron EC-targeting via the ROB04 enhancer promoter. 2) Constitutive expression of LUC for BIX bone metastases growth, inhibition, or recurrence detection and EGFP for enhanced tissue

immunofluorescence localization, 3) Constitutive prodrug converting enzyme expression that is functionally conditional due to prodrug dependence, and/or 4} Switchable doxycyeline control of multiple stem cell ligand decoys.

Testing Ibr CSC/HSC/HPC mobilization can be implemented. Combinatorial S-F€:5- FU generation with mu!ti-ligand mediated CSC niche eviction can be tested for metastatic growth inhibition efficacy compared to solo Ad~bCD vector data. Vector pretumor injection treatment can allow us to perform tumor dormancy and established tumor experiments using a single experimental design. Experimental duration can be extended and sequential vector polycisiron expression acti vation performed on recurrent tumors of selected sixes.

imaging experiments can test metastatic tumor burden detection thresholds. To probe transktional relevance our vectors can be tested for prolonged expression in syngeneic bone metastatic models. Additional viral, capxid genetic and possibly chemical engineering can also be engaged obviating the anti-coagulant factor and producing immune evasion. Gene fusion strategies, viral species/type of 2 A peptide and cDNA cassette pofycistron ordering can all be altered to achieve a polycistronic vector requisite for bone .metastatic efficacy. The .numbe of cistrons can be reduced to achieve a functional encapsidated vector.

Example 18

This example illustrates that MBP pseudo-typing attenuated hepatoeyte vector expression while producing widespread multi-organ vascular EC expression.

The ultiorgan biodisirifeution of Ad.MBP.CMV was tested using semiquantitative tissue section immunofluorescence analysis. AdS.CMV-mediated expression, was predominantly localized in Iiver hepatocytes and detectable in reticuloendothelial system and endothelial cells (ECs) of spleen (PIG. 28A). Vector expression was scarcely found in lung, heart, kidney, gastrocnemius muscle, pancreas, small bowel, large bowel, and not detectable in any part of the brain (FiG. 23B). In contrast, Ad.MBP.CMV produced EC expression throughout the mtcrovasculature of heart kidne , muscle, pancreas, intestine, and brain (FiG, 23A). Vector EC co-localization was confirmed using high-magnification EFT imaging in these organs (FiG. 29). Surprisingly, robust iransgen.e expression was detectable in BCs within tested brain regions including cerebrum, cerebellum, hippocampus, and medulla (FIG. 28B). To quantify vector transgerte expression, EGFP fluorescence intensity was summed in a tissue region of interest (RQ1) and normalized by the ROl are (per pnr) in each organ. Liver sections from Ad. BP.CMV-mjected mice exhibited a 5-fold reduction in the EGFP fluorescence intensity compared with the AdS.CMV counterparts (FIG.23B). Liver

detargetlng was associated with 2-fold increase in vector expression in splenic

reticuloendothelial cells and ECs (FIG. 23.8). As long, heart, kidney, pancreas, small and large bowel and brain were eithervbarely or not transdueible by the AdS.CMV, the retargeting enhancement of the Ad.MBP.CMV to these organs was ranged, from greater than 10-fold increase in pancreas, small bowel, and. large bowel, greater than 100-fold increase in lung and kidney, greater than 1, 000-fold increase in heart and muscle, and greater than

10,000-fold increase in brain (FIG. 238, red bars versus blue bars for Lu, H, K, M, P, SB, LB. and B).

FiG., 28 illustrates incorporation of M.BP into Ad5 detargeted the virus from liver hepatocytes, modestly increased gene expression in splenic marginal zone, and markedly enhanced gene expression in all regions of the brain. (A) EGFP expression in liver and spleen following intravenous injection of xlO¹⁵ v of AdS.CM V or Ad.M^'BP.CMV into adult

CS7BL/6J mice, AdS.CMV expression was widespread and robust in liver hepatocytes (top left panel) and punctate within splenic marginal zone (top right panel). A&MBP.CiVIV markedly reduced vector expression in liver hepatocytes (bottom left panel) with increased vector targeting to splenic marginal zone (bottom right panel). Co-staining of spleen sections with EC markers endomucin and C 31. indicated that Ad.MBP.CMV was targeted to mixed ECs and other cell popul tions), (B) immunofluorescence microscopy analysis of EGFP expression in difTerent region of the brain following intravenous injection of 1x10'* vp of Ad.MBP.CMV into adult C57BL/6J mice. EGFP expression was widespread throughout the vascular network of the cerebrum, hippocampus, medulla, and cerebellum. Magnification: 100X, Red: endomucin CD3l, Green: EGFP immunofluorescence. Blue: DAPL

FiG. 23 illustrates incorporation of M.BP into AdS drastically increased viral gene expression to vascular beds of multiple host organs. (A) Immunofluorescence microscopy analysis of vector EGFP expression in host organs following intravenous injection of 1x10^U viral particles (vp) of Ad.MBP.CMV raio adult CS7.BL 6J rake revealed pr6.rai.nent transgerie ex ression in lung, heart, kidney, gastrocnemius muscle, pancreas, small and large bowel, and brain, Co-staining of tissue sections with an EC-specific endomucm D31 cocktail revealed that EGFP expression was restricted to the vasculature. (B) EGF fluorescence per pro² of tissue section area (FL tluorescence intensity) in each, organ derived from AdS.CMY- ffijeeied mice (tv"^:4 for all organs) versus thai from Ad. M.BP.CMV ojected mice (n^∞10 for liver, spleen, heart, kidney, muscle, small bowel, and brain; -l for lung, pancreas, and large bowel), (C) The percentage of vascular EC area expressing EGFP in each organ deri ved from AdS.CMV'-injected mice (η·""4 for all organs) versus that from Ad. BP.CMV-injected mice (n-- 0 for heart, kidney, muscle, small bowel, and brain; IF^::7 for lung, pancreas, and. large bowel). Bar graph is mean A standard deviation asterisk: adjusted p<0.0S, Magnification: !OOX, Red: endomucin/Q>3 L, Green: EGFP immunofluorescence. Blue; DAP1. Li: liver, S; spleen, Lu: lung, H: heart, : kidney, M: muscle, P: pancreas, SB: small bowel, LB: large bowel, B: brain.

The EC-expression efficiency of the Ad.MBP.CMV versus AdS.CMV in multiple organ was determined by quantifying the percentage of total tissue EC area expressing EGFP in each organ. Ad.MBP.CMV targeted greater than 63% of blood vessels in regions of the brain (B), 2.1% in lung (Lu), 26% heart (H), 33% in kidney ( ), 3B% in muscle (M). 30% in pancreas (P), 16% in small bowel (SB), and 6% m large bowel (LB) {PIG, 23C). Other than liver and spleen, pancreas and small bowel were the onl detected organs where

AdS.CMV produced an appreciable but still rare vascular EC expression (FIG. 23C).

To furthe test EC-specific expression, immunofluorescence for the pericyte markers, PDGFRp¹ or proteoglycan nerve-giiai antige 2 (NG2), and the pan-heraatopoietic lineage cell marker CD45 or macrophage marker F4 80 were performed. High-magnification revealed that the EGPP~esp.ress.ing cells were distinct from the PDGFRp-posirsve or NG2-positive ceils in tested organs (FIG. 29). Ad.MBP.CMV was expressed in rare CD 5-posiiive hematopoietic cells and F4 80-positive macrophages in liver and spleen, but not in any other sampled organs (F G . 30).

FIG. 29 illustrates Ad.MBP.CMV selectively targeted vascular PCs but not pericytes in multiple host organs. High-power magnification EFT (Methods) micrographs of tissue sections co-stained with an endomucin/CD31 cocktail (top panels) and an EGFP antibod localized Ad.MBP.CMV transgene expression to vascular EC's by the in lung, heart, kidney, muscle, small bowel, large bowel, and. brain. Tissue sections co-stained for vascular pericyte marker PDGFRp (middle panels) or proteoglycan nerve-gliai antigen 2 (NG2, bottom panels) revealed that the EGFP-expressing cells in the organs were distinct from the PDGF.R$ or NG2+ cells. Magnification: 4G0X, Red: CD31/endomucin for top-row panels, PDGFRp for middle-row panels, and NG2 for bottom-row panels. Green: EGFP immunofluorescence, Blue: DAPL

FIG. 30 illustrates Ad.MBP.CMV targeted ceil populations) distinct from CD45- positive or F4/80-positive cells in most host organs. High-power magnification EFi micrographs of tissue sections co-stained for EGFP and hematopoietic cell marker CD45 or macrophage marker F4/80 in eight-organ panels. The EGFP-expressing cells were distinct from the CD45-^;- hematopoietic ceils and F4/80 macrophages in Hmg, heart, kidney, gastrocnemius muscle, small bowel brain. However, a small fraction of EGFP-positive-ceUs in liver and spleen expressed CD45 and F4 S . Magnification: 400.X. Red: CD45 for top-row panels and F4/80 for bottom-row panels. Green: EGFP immunofluorescence. Blue: DAP!.. Example 19

This example illustrates that warfarin liver detargeting failed to increase Ad.MB^'P.CMV niuiiiorgan EC expression.

While the Ad.MBP.CMV yielded an impressive level of hepatocyte detargeting, liver remained a substantial transdueticnal and transcriptional target (FIG. 24).. As the major pathway directing AdS hepatocyte sequestration is. mediated by coagulation Factor X-viral hex on binding, it was tested whether warfarin could affect diminution in the level of

Ad.MBP.CMV expression in hepatocytes. (Waddington, S.N._< et al. 2008) Warfarin

pretreatment diminished the number of EGFP-expressing hepatocytes (FIG. 24 A). The residua! number of vector expressing hepatocytes was similar to our previous work w ith AdS based vectors with wild type capsids. This residual, hepatocyte vector expression following warfarin treatment has been seen by others and likely represents "floor" for die efficacy of pharmacoiogicai blockade. (Waddington, S.N., et-ai. 2008) However, in contrast to our previous work with vectors with wild type capsids, (Lu, Z.EL et al 2-013) warfarin failed to enhance EC expression in the other testeclorgans (FIG. 24A and FIG. 28), These data suggested that either the Ad.MBP.CMV peripheral vascular EC vector expression was saturated at our 1 x.KF ^f viral particle dose, or that complement opsonization facilitated destruction of the vector dose increment that escaped hepatocyte transduction. (Xu, Z., et al 2013).

FIG. 24 illustrates that warfarin pretreatment reduced Ad.MBP.CM V liver tropism but did not alter gene expression in other host organs. (A) Warfarin, 5 mg/kg, on day -3 and - .1 before vector injection diminished hepatocyte expression but did not change transgene expressi on in spleen. (B) EGFP fluorescence per μ«τ of tissue area in each organ derived from warfarin-treated mice (rHJ for ail organs) normalized as percentage of the mean value of vehicle- treated or untreated counterparts (nHO for liver, spleen, heart kidney, muscle, small bowel, and brain; n^m7 for lung) with standard deviation. Warfarin pretreatment reduced vector liver expression by 68% (Li) but did not lead to a significant change in gene

expression in spleen (S), long (Lu), heart (H), kidney ( )_y muscle (M), small bowel (SB), or brain (B). Asterisk indicates adjusted p 0.05. Magnification: 100X, Red: C.D3 t/endoraucin. Green: EGFP immunofluorescence, Blue: DAP!

Example 20

This example illustrates that Ad.MBP.CMV dose reduction produced organ-specific iion-!inear EC expression reduction.

In these experiments, mice were challenged with injection of 2xl 0^!i! viral, particles to test the sensitivity of each organ vascular bed for Ad.MBP.CMV expression. The lower viral dose reduced tissue Ad.MBF.CMV expression in liver, spleen, pancreas, heart, kidney, muscle, pancreas, small bowel and brain. Frequency and EC expression level, in the lung remained unaffected by vector dose .reduction {FIG. 25A and FIG. 25B). Comparison of EGFP fluorescence intensity of low- versus high-dose tissue samples revealed that splenic and brain transgene expression was 16% an d 31 % of the high-dose counterparts (FIG. 25C, S and B). These levels of reduction in. EGFP expression were within the range of linear response to the viral dose difference (20%). However, the diminished expression in liver (5% of high-dose level), heart (0.4% of high-dose level), kidney (0.5% of high dose level), muscle (0.1% of high-dose level), pancreas (0.4% of high-dose level), and small bowel (3% of high- dose level) wa nonlinear. Similar to EC expression frequency analysis, vector dose reduction failed to significantly dimi n ish transgene expression in Umg (91% of high-dose level, p^;::0.5S8). These results .show that Ad.MBP.CMV lung-specific EC expression targeting .may be achievable through vector dose fine tuning.

F G. 25 illustrates systemic administration of a low dose ofAd.MBF.CMV into adult mice produced differential and non-linear reduction in. gene expression in host organs. (A) EGFP expression in host liver, spleen, lung, and brain following intravenous injection of I x l 0'ⁿ or 2x10^Ui vp of Ad.MBP.CMV into adult mice. Lowering vector dosage significantly reduced EGFP expression in vascular BCs of liver, spleen, and brain but did not change the expression in Sung. (B) EGFP fluorescence per μαι of tissue area in each organ deri ved from the low-dose grou (n^;;;6 for each organ). (C) Normalization of the tissue EGFP fluorescence intensity values in (B_.) to the mean value of the high-dose counterparts. The spleen and brain EGFP expression in low-dose group was 1 % and 31% of the high-dose counterparts.

However, low virus dose drasticall diminished the transgene expression in, heart, kidney; muscle, pancreas, and small bowel (3% of high-dose level). The low dose did not significantly alter the transgene expression in lung (91% of high-dose level). Asterisk indicates p<0.05. Magnification: 1 QOX, Red: endot«ucin€D31 , Green: EGFP

immunofluorescence. Bloc: DAPE Li: liver, S: spleen, Liv. lung, H: heart, : kidney, M: muscle, P: pancreas, SB: small bowel, B: brain.

Example 2 !

This example illustrates that mononuclear cell depletion failed to di minish

Ad.MBP..CMV EC transgene expression.

The Ad,MBP.CM V acquired a specific and high affinity binding to myeloid cells ex viva, compared with the Ad5. (Alberti, MO., et !. 2013; Alberti, M.O., et al. 2012).

in these experiments, two daily doses of intravenous clodronate liposomes were administered to test the necessity of mononuclear cells for Ad.M.BP.CMV EC transgene expression iti intact mice. The percentage of circulating CD1 lb-positive cells and the level of inuki-organ tissue section EGEP fluorescence was measured. (FIG. 26A~ FIG. 26C), Clodronate treatment depleted circulating CD1 lb-positive leukocytes by 77% and completely depleted F4/80-positive macrophages in. liver (Kupffer cells) and spleen {^'FIG. 26A. and FIG. 3.1). in contrast, clodronate barely reduced resident macrophages in lung, small bowel, heart, and kidney (data nor shown). Clodronate increased Ad.MBPXMV EC lung expression by 2- d but did not significantly alter EC transgene expression level or the EC-specific expression pattern in liver, spleen, heart, .kidney, muscle, pancreas, small bowel, or brain (FIG, 26B). The lack of increase in. hepatocyte was surprising given prior reports on the scavenging function of liver Kupffer ceils (Wolff, G,, et a!, 1997) however, others have also reported a modest, though sta tistically insignificant level of clodronate-medsaied Ad vector liver expression enhancement. (Bradshaw, A.C, et al 2012) These data demonstrate that circulating monocytes and macrophages are dispensable for Ad.MBP.CMV organ EC expression. However, the persistence ofextrahepatie organ Cdl lb and F4 8G cells following clodronate depletion does not rule out the marginaled myeloid cell pool as a mechanism for vector-EC hando.f (Alberti, M.O., et al. 2013).

FIG..26 illustrates depletion of circulating monocytes and hepatic and splenic macrophages lead to an increased Ad.M.BP.CMV^' gene expression in the lung without a significant change in gene expression in other organs. (A) Representative flow cytometry plots (left panel) quantifying the FSC-htgh/SSC-low/CD? 1 b-positi-ve;C 5-positlve monocyte population in circulation.. Relative frequency (right panel) of circulating monocytes from clodronate liposome-treated mice (clod, n~3) versus saline-treated mice {veh, rrt). Intravenous injection of clodronate liposomes depleted circolatiog CD! 1 h-positive cells by 84%. (B) EGFP fluorescence per pm² of tissue area in each organ derived from Uie saline- injected mice (n^∞ 7 for liver, spleen, heart, kidney, muscle, pancreas, small bowel, and brain; n™4 for lung) versus clodronate liposome- injected mice (tH$ for liver, spleen, heart, kidney, pancreas, small bowel, and brain; n-7 for muscle; n- 5 for lung). Intravenous clodronate increased Ad.MBP.CMV lung expression by 2-fold (Lu) but did not result in a significant change in gene expression in liver (Li), spleen (S), heart (H), kidney ( ), muscle (M), pancreas (P), small bowel (SB), or brain (B). Asterisk indicates adjusted p<(),05.

f H i 3 ! illustrates depletion of hepatic and splenic macrophages by clodronate liposomes. Micrographs show F4/80 expression in liver and spleen from saline-treated mice (veh) or clodronate iiposome-treated mice (clod). Clodronate-liposome treatment completely depleted F4 80~positive macrophages in liver ( op ler cells) and in spleen, red pulp region. Magnification: iOOX, Red: B4/80, blue DAPI.

Example 22

This example illustrates that EC-specific ROB04 gene promoter/enhancer ablated the MBP vector hepatoeyte expression but reduced EC expression in other organs,

in these experiments, an AdS vector was engineered for transcriptional targeting of ECs using the EC-specific human ROB04 gene enhancer/promoter fragment ( aliberov, S.A., et al. 2013; Lu, 2., et al. 2013) The CMV promoter was replaced with she OB04 enhancer/promoter to test whether the combination of transcriptional with transducfional targeting could produce enhanced multi-organ EC expression. The dual targeted

Ad.MBP,ROB04 vector was administered, intravenously and organs were analyzed for vector transgene expression (FIG. 27 A). Ad.MBP..R.OB04 abrogated hepatoeyte expression and instead EGFP was detectable in a scattered population of liver ECs (FIG. 27A). The

Ad.MBP.RQB04 also produced EC transgene expression in an appreciable vascular area fraction in spleen (23%), kidney (23%), lun (10%), muscle (9%), beast ( 10%), and brain ( 15%) but produced very low expression in small bowel, and large bowel (I % and 2% respectively) (FIG. 27B, S, Lu, H, K, M, SB, LB, and B). Collectively, the ROB04 enhancer/promoter produced a lower host organ EC expression compared to Ad.MBP.CM V in each organ. However, the undetectable vector transgene expression in hepaiocytes highlighted the enhanced endothelial cell type stringency of the D804 compared to the CMV promoter in the Ad.MBP vector. PIG. 27 illustrates Ad,MBP.ROB04 detargeted hepatocyte expression but reduced levels of vascular EC expression in other host, organs. (A) EGFP expression following intravenous injection of I x U)^u vp of Ad.MB P. ROB0 into adult mice . Ad.MBP. ROB04 yielded punctate vascular EC expression in liver but showed a reduced targeting efficiency to vascular ECs in spleen, lung, heart, kidney, muscle, small bowel, and brain. (B) The EGFP- positive vascular area analysis was performed as shown in FIG, 23C. Magnification: !OOX, Red: endornuem/CD3 i , Green: EGFP immunofluorescence. Blue: ΌΑΡί, Li: liver, S: spleen. Lie long, H; heart, : kidney, M: muscle, SB; small bowel, LB; large bowel, B: brain.

Example 23

This example illustrates Ad.MBP .ROB04-EGF.P and Ad.RGf>.ROB04-EGFP expression in In farcy Repermsion (I/R) and non-l/R regions.

In these experiments, mice were subjected to suture induced left anterior coronary artery iseheinia/reperfusion. One day later, either A&MBP.RQB04-E FP or

Ad.R.GD.ROBQ4-EG.PP was injected intravenously.. The left ventricle evidenced injury as evidenced by monocyte infiltration. (FIG, 32A and data not shown), frank infarction (FIG. 32D), and angiogen.es.is (arrowheads in FIG. 32A and FIG. 32D). Both vectors were expressed in the I/R region, Ad.MBP. OB04-EGFP was induced as indicated by the green EGFP immunofluorescence (FIG. 32 A) tn the 1/R region, whereas it was expressed in multiple vessels in other heart regions not subject to I R, but at a lower level (FIG. 32B and FIG, 32C). In contrast, Ad.RGD.ROB04-EGFP expression was restricted to the I/R region (FIG. 32Ό), albeit at a lower level than Ad.MBP. /RfTB04~FGFP, Ad. RGD , ROB04- EGFP was not expressed in non-l/R regions such as the left ventricular septum or the right ventricular wall (FIG. 32E and FIG. 32F respecti ely).

Example 24

This example illustrates uses of the Ad.MBP platform to enhance and/or facilitate limb salvage.

to these experiments, both Ad.MBP.CMV (Lu et ai. 2014) and Ad,MBP.ROB04 vectors can be induced in the vascular endothelium of the adductor skeletal muscle following hiiidiimb ischemia secondary to femora! artery ligation in a mouse (PIG. 33), Vectors using an Ad.MBP platform can be loaded with tratisgene(s)-expressed secreted angiogenic and arteriogenic -growth factors and/or transcription factors such as constitutive HIF l -alpha (Ola lipupo et al. 2011), HlF2-alpha mutants and/or other master regulatory transcription factors. These factors can have the ability to coordinateiy induce suites of gene targets mediating a plethora of molecules that can. enhance and/or facilitate limb salvage in the context of atherosclerotic disease alone or as a consequence of diabetic vasculopathy.

Example 25

This example illustrates uses of Ad. BP vectors to treat conditions activating angsogenesis in villous endothelium,

Ad.MBP vectors are expressed in small and large intestinal vascular endothelium (FIG, 34) (Lu et al. 2014). Ad vector Ad.MBP.R0BO can be specifically induced in angiogenic intestinal villous vascular endothelial eelis following massive small bowel resection, in contrast to a lack of expression in sham-operated small bowel An

Ad.MBPJl0604 vector can be specifically expressed in other conditions activating angiogenesis in villous endothelium such as the inflammatory bowel diseases regional enteritis and inflammatory bowel disease of the colon, infections with toxin producing bacteria such as Clostridium difficile, Clostridium botulinum, Shigella, and- in the colon cancer precursor lesions of multiple polyposis. Intestinal vascular-trophic vectors can be armed with transgenes that produce secreted anti-inflammatory cytokine decoys such as soluble T F-alpha receptor, or single chain anti-lL l lLI 7 antibodies, bacterial anti-toxins, and RNAi molecules targeting gene products induced by the activation of t he NT pathway in multiple polyposis.

Example 26

his example illustrates use of an Ad.MBP.CMV vector to treat inflammatory diseases and degenerative diseases.

The Ad.MBP.CMV vector can be expressed in ail regions o f the brain (Lu et aL 201 ), This diffuse expression pattern can be used to produce secreted proteins engineered to cross the blood brain barrier and designed to treat inflammatory diseases such as amyotrophic lateral sclerosis and multiple sclerosis and degenerative diseases such as Alzheimer ^*s and Parkinson's. (FIG. 35), For primary and metastatic brain tumors in particular, an

AdJR.GD.H5/H3 vector was specifically expressed within the metastatic vasculature but not in normal brain vasculature (FIG. 36B and FIG, 36C). Data also demonstrated expression of the Ad.MBP.CMV vector in the brain vasculature surrounding the hypothalamus (FIG. 35). Aft A&MBP.CMV vector can be engineered to express secreted molecules affecting the hypothalamic appetite nuclei (arcuate). Vectors, such as the vectors in this example, can be used to stimulate appetite in patients suffering from cachexia either due to cancer or benign conditions, or to induce satiet in obese patients with the metabolic syndrome.

Example 2? This -example illustrate use of Ad.RGDlfS./H3,ROB04 ant! parental A&R.0804 vectors to treat cancers, produce anti n.fiaraniatory molecules to treat rnyelodysplastic syndrome bone marrow, and/or correct genetic diseases.

The Ad.RGDTi5/l .ROB04 and parental Ad.RO.B04 vectors are expressed throughout the sinusoidal endothelium of the bone marrow (FIG. 37). These vectors can be engineered to express secreted molecules that can mobilize metastatic cancer or leukemic stem cells from their protected niches for chemo-irradiation sensitization (such as molecules described in Nervi et al. 2009), kill metastatic cancers due to ehemotherapeutic- prodrug converting enzyme production (such as molecules described in Guyasg et al, 201 i), to produce anti-inflammatory molecules to treat rnyelodysplastic syndrome bone marrow, and/or correct genetic diseases such as hemophilia and sickle cell anemia. FIG. 38 demonstrates expression of Ad.RGD.H5/H3.ROB04 within the vasculature of metastatic human prostate cancer in the femur of a mouse.

Example 28

This example illustrates that the angiocrine function of endothelial cells can he- manipulated using vascular targeted adenoviral vectors.

The vascular endothelium can be engineered to secrete molecules that can affect the vascular endothelium's local microenvironments either in tumors or benign diseases,.

Angiocrine function is the term for the concept of vascular endothelium regulating its mieroenvironraent via molecular secretion. Regarding Ad.ROBO4-bC0 (bacterial cytosme deaminase enzyme), tor example, the cytosme deaminase enzyme converts the inactive prodrug S-fluorocytosine (S-FC) to cbemotherapeutic, 5-fluorouracil t 5-FU).

Mice were warfarinized because this drug prevents liver sequestration of the Ad.ROBfM vector (not necessary with Ad,RGD.H5 H^'3 or Ad. BP vectors FIGS. 32-37 above), and administered 5-FC for 12 days. There was a focal -ablation of the bone marrow hematopoietic elements presumably due to 5-FU production and secretion by adjacent vascular endothelial cells. The non-dividing blood vessels were preserved, albeit dilated, despite destruction of the adjacent hematopoietic cells (FIG. 39).

These data demonstrate that the angiocrine function of endothelial cells can be manipulated using vascular targeted adenoviral vectors including vectors listed in this example.

FIGS. 32A-32F illustrate induced expression of Ad.MBP. OB04-EGFP and.

Ad.RGIXROB04-.EGFP vectors in region of isc-hemia-reperfusion (f/R.) in a suture mouse model. FIG. 32A illustrates Ad.M.8P.ROB04 expression, in the left ventricular 1 R region. FIG. 328 illustrates Ad.MBP,R0BO4 expression in left ventricular septum, FIG. 32C illustrates A& BP.ROB0 expression, in. right ventricular free wall. FIG. 32D illustrates Ad.RGD.ROB04 expression in left ventricular !/R region. FIG. 32E illustrates

Ad.R0D, 0BO4 expression m left ventricular septum. FIG. 32F illustrates

A&RGD.ROB04 expression m right ventricular ftee wall Red; vascular endothelial specific immunofluorescence using a CD3 i e«do mucin antibody cocktail. Green: EGFP

immunofluorescence. Blue: DAP! nuclear stain. Magnification: 40X.

FIG. 33 illustrates Ad.MBP.ROB04-BGFP expression in the vascular endothelium of the adductor (thigh) muscle following hindlimb ischemia secondary to femoral artery ligation. Red. Green, Blue as in FIG. 32. Mag: 40%.

FIGS. 34A-34C illustrate adenoviral vector expression localized within angiogenic villi in a small bowel resection (SBR) model. FIG. 34A illustrates mice injected with.

Ad.MBP.ROB04-EGFP five days post sham surgery. FIG. 34B illustrates endothelial and possible lymphatic expression of the same vector in angiogenic villi post SBR. FIG. 34C illustrates high power view of villous in FIG. 34B (arrowhead) showing eotoealized vector iransgene expression in angiogenic sprouting endothelium (arrowheads indicate sprouts). FIG. 34A and FIG. 34B 100X, FIG. 34C 400X.

FIG. 35 illustrates Ad. BF.CMV vector expression to. the vascular endothelium surrounding the hypothalamus (encircled). Red, Green, Blue as. in FIG. 32. Mag: 40X:.

FIGS. 36A-36C illustrate expression of Ad..RGD.H5/H3 vector within the vascular endothelium of human prostate brain metastases in a mouse. FIG. 36A illustrates a histological section, that is adjacent to FIG. 36B. FIG. 36C illustrates a prostate brain metastases in another mouse. Asterisks denote metastases, cross uninvoived brain. Red,, Green, Blue as in FIG. 32. Mag; iOOX.

FIGS. 37A-37B illustrate Ad.RGD.H5/M3.ROB04 vector expression in bone marrow sinusoidal endothelium. FIG. 7A illustrates cortical bone marrow in bone shaft. FIG. 37B illustrates trabecular bone marrow near bone end and cartilaginous plate. Red, Green, Blue as in FIG. 32. Mag; I OOX,

FIGS. 38A-38B illustrate expression of Ad.^'RGD.R.OB04~£GFP in a iGR-CaPl human prostate cancer femoral bone metastases in OD/SC1.D/IL2RY immunodeficient mouse. FIG. 38A illustrates an adjacent section to FIG. 38B. Green and yellow asterisks are hematopoietic cells adjacent to metastasis. White and black asterisks are de novo, osteoblastic bone. White and black crosses are metastatic ceils. Arrowhead delineates osteoblastic "rimming", a pathological hallmark of osteoblastic metastases.. Red, Green, Blue as in FIG. 32, Mag; IOOX. FiGS. 39A-39D illustrate angiocrine production, of S-iluoro uracil (S-FU) from bone marrow sinusoidal endothelial cells expressing eytosine deaminase (bCD) from an

Ad. OB0 vector. FiGS. 39A-39D illustrate bone trabecular histology from a mouse injected with Ad. R.OB04-EGFP control virus. FiG, 39B illustrates corresponding^' vascular marker immunofluorescence. FIG. 39C illustrates bone trabecular Iiistopathology 5-FC treated mice following Ad. .OB04-bCD and preinjection warfarin to detarget liver

hepatocyte vector sequestration. FIG, 39D illustrates vascular immunofluorescence

demonstrating dilated but intact vasculattire and apoptoiie hematopoietic ceils. Red and Blue as in FIG. 32. Mag: 100X.

All references cited herein are incorporated by reference, each in its entirely.

Applicant reserves the right to challenge any conclusions presented ^'by the authors of any reference.

References cited:

Aird, W,C. Endothelial hiomedkine, Cambridge ; New York; Cambridge University Press,

2007, p. xl 1856 p., 1824 p. of plates.

A! Nakoiizj .„ et al. Neoplasia 2012; 14:376-387.

Alba R.. et aS. Blood. 2010: 1 16:2656-64.

Alberts M.O.. et al. Gene her 2012,

Alberts M.O., et al. PLoS 0ne 2O 2;7;e378I2.

Alberti, M.O., et al. <ie therapy 2013 20(7):733-74L Epub 2012 Nov 22.

Assi, H,, ei al. Ne rosvimce tetters 2012;527(2}:?l -77.

Azuma M., et al. Biochem Biopbys Res Comtnun 200S;338; .l 164- 1 170.

Bachtarzi H„ et al. J Control Release, 201 1 ; 150: 196-203.

Bachtarzi, K, et al. Journal of drug targeting 201 .1 ;19(8}:690- 700.

Baker All., et al. Exp Physiol 2005:90:27-31.

Barker N., et al. Cell Stem Cell 2010;7:656-670.

Bedell V.M., et al Proc Natl Acad Set U S A. 2005; 102:6373-8.

Beiousova, N., et al Journal of Virology 2003;77(2 i); H 367- 1 .1377.

Bergers G._s et al Nat Rev Cancer. 2008;8:592-603.

Borghese C\, et al J Cell. Biochem 2013; ! 14: 1 ! 35-1 344.

Bradshaw, A.C., et al. Journal of controlled release: offickd journal of the. Controlled Release Society 2012;164(3);394-402.

Bradshaw, A.C., et al Vascular pharmacology 20!3:58(3):174- 18i . Brenner A,, et al. Clm Cancer Res 2013.

Butler JM, et al. Nat Rev Cancer.2010;10:138-46.

Carey BW, et al. Proc Natl Acad Sci U S A 2009;106:157-162.

Chang HL et al. ,1 Biomed Sci 2011:18:6.

Chauchereau A, et al. Exp Cell Res 2 11 ;317:262-275.

Chung L W , et al. Bone and Cancer. F. Bonner and M. C. Faracn-Carson. London, Springer- Veriag.2009.5: 157-166.

Cole, C, et al. Nature medicine 2005; 11(10}:! 073-1 8 ! .

Cook KM, et al. CA Cancer J Clin.2010;60:222-243,

Co«ne_: P.O., et al. ('old Spring Harbor perspectives in medicine 2 l2;2(4);a0094 1,

Crawford Y, et ai. Cancer Cell.2009; i 5:21-34.

Ding L, et al. Nature 2013;495:231-235.

Ding L, et al. Nature.2012;483 ;457-462.

Dong Z, et al Adv. Drug DeUvRev.2009;61:542-553.

Duarte S, ct al. Cancer Lett 2012;324:160-170.

Dubrovska A, etal. PLoS One 2012;7:e31226.

Duffy M , et ai. Nanomedicme (Load).2012;7;271-288.

Economopouiou , Nat. Med 2009;! 5:553-558.

English HF, et al. Prostate 1 87; 11:229-242.

Essers MA, et l. Mol Oncol 2010;4:443-450.

Falko ska-tfausen B, et ai. Exp Cell Res 2010;31 : 1885- 1 95.

Ferrara N. Cur? Opiit Hematol.2010;17:21 -24.

Ferrara N. Eiiioct. Rev.2004:25:581-6 1,

Ferrer-Vaquer A, et al BMC Dev Biol 2010:10:121.

Foudi A, et ai. Nat Bioiechnoi 2009;27:84-90.

Frite V, et al J Cell Biocfaera 2 11 ;112:3234-3245.

Fuchita M, et al Cancer Res 2009;69:47 1-4799.

Funahashi Y, et ai. Cancer Res 2008;68:4727-473 .

Galan-Moya, EM., etal. MBO reports 2011;12(5):470-476.

Glinsky VV, Cancer Metastasis Rev 2006;25:5 1-540.

Goldman CK, et al. Proc Nat! Acad Sci U S A.1998;95:8795-8800.

Goldstein AS. et al.. FM.BO Rep 2012;13:1036- 037.

Gordan JD, et af. Cancer Ceil 2008;14:435-46.

Greenbaum A, et al Nature 2013;495:227-230. Greenberger S, et al. J Clin invest.2004:113: .1017-1024.

Guo C, et al Methods Mol Biol 2012:8/9:315-326.

Ma.djaiHon.akis AK, etal Nat Rev Genet 2003;4:613-625.

Hadjaotonakis AK, et al, BMC Bi technol 2004:4:33,

Haistna 10, etal M i Pharm.2010:391:155-16! .

Hatfield 3, et al Biochem Pharmacol 2011:81:24-31.

He, T C, et ai. Proceedings oft.be National Academy of Sciences of the United Sta-

America 1998;9S(5);2509-2514,

He Y, et al Gene Expr 2010;15:13-25.

fieidenreieh R„ et al. Cancer Res 2000:60:6142- 6147.

Hirbe AC, etal. Proc Natl Acad Sci U S A 2007;104:14062-14067.

Horvath LG, etal Prostate 2007:67:1081-1090.

Haminiecki L, et al. Genomics.2002;79:547-52.

Ibrahim T. et al. Cancer 2010;!.] 6: 1406- 41 S.

Jaggar RT, et al. Hum Gene Ther 1 97;8:2239-2247,

Jones CA, et al Nat Med.2008;14:448-53.

Jones CA, et al Nat Ceil Biol 2009; ! 1:1325-31.

KaUberov SA, et al Cancer Gene ^"flier 2006;13:203-214.

KaUberov, S.A., et a!. Wrohgy 2013; 47( 1 -2):31 -325.

Kamdar, F., el ai. Canadian journal ofphyx/o g}' and pharmacology 2012;90 SO): 1 1344.

Kand T, etal Curt Bio! 1 98;8:377-385..

Karhadkar SS, et al Nature 2004;431:707-712.

Kaminsky, S.M., ei al. Human gone therapy 20!3;24{ 11};948-963.

Kawano Y, et al Br J Cancer 2Q0 ;100:1165- f 174.

hare R, et a!. Curt Gene Ther.201 ! ; 11:241-58.

Kiel et al Ceil 2005;121:1109-112

ievit E, et al Cancer Res 2000;60:6649-6655.

Kim ill et al Proc Natl Acad Sci li S A 2001 13282-13287.

Kim il et al Clin Cancer Res 2012.

Kobayashi H, et al Nat Ceil Biol 2010; 12: 1046-1 56,

Koch AW, et al Developmental Cell 20! l;20:33-46.

Koh Bl ei al EMBO Rep 2012;13:412-422.

Kondo , et al FloS Biol.2003;! ;E83. osteraiik PJ_> et a!, J Bone Miner Res 2009:24; ! 82-195.

Lavergne E, ei ai. Oncogene 2011;30:423-433.

Leenders WP, et al Clin Cancer Res.2004;10:6222-30.

Leong KG,etai. Differentiation 2008;76:699-716.

Li, C, et al Proceedings of the National Academy of Sciences of the United Staies of

America 20.11 : 1 8(34): 14258-1 263.

Undername IX ei ai. Thromb Maemost.2009;102:1135-1143.

Logan M, et al Genesis 2002;33:77-80.

Lu, Z.H., etal WoS One 2013;8( 12):e83933.

Lu Z.H., et a!., Lab Invest 2014;in press.

Lukacs R.U, et al. Nat Protoc 2010;5:702-713.

Mahasreahti PJ_t et al. Clin Cancer Res 2001;7:2057-2066.

Marignoi L, etal. J Gene Med 2009; 11:169-179.

Marlo R. et al Proc Nail Acad Set U S A.2010;107:10520-5.

Mavria G, et al. Gene Ther.2000;7:368-76.

Mayle A, et al Cytometry A 2013;83:27-37.

Miki 3, etal Cancer Res 2007;i>7:3153- 161.

Mognetti .8, et al. j Cell Mol Med 2013;17:287-292.

orrissey C, et al Clin Exp Metastasis 2008;25:377-388.

Muhammad AK, et ai. Clin Pharmacol Ther 2 10 $8:204-2.13.

Muro, S., etal Current wmtfetr phamacofogv 2004:2(3):281-299.

Nagasa a T, et al Trends Immunol 2011;32:31 -320.

Nai ani AC, et ai. N Engl J Med 20l1.;365:2357-2365.

ervi B, et al. Blood 2009; 113 ; 206-6214.

Netteibeek DM, et ai. Mol Ther.2001;3:882-891.

owotsc m S, et ai. BMC Dev Biol 2013;13: 15.

Okada Y, et al Blood.2008:112:2336-9.

Okada Y, et al Blood 2008;! 12:2336-2339.

Okad Y, et al . Cire Res.2007; 1 0: ] 712-22.

Oiadipupo, S.S., et ai. Mfood liH 1;1 l?(i5}:4.142-4153.

Omatsu Y, et ai. Immunity 201 ;33:387-399.

Ou, D.B., et al PloSone 2013;8(l):eS5233.

Ouyang, L, et al,. Bioorg Med Chera 20! 1 ;1 { 12}:3750-3756.

Park W, et ai. Dev Biol.2003;261:251-67. Pe!ed M, et al. Clin Cancer Res, 2009;15:1664-73.

Preuss MA, et al. Open Gene Ther J. 2008; ! : 7-1 i .

Qin J, et al. Cell Stem Cell 2012; 10:556-569.

Ra aswamy, S.₅ et al. Neurobiology rfd tm 2012:48(2 ):243-2S4.

Raper SB. et al. Mo) Genet Metab 2003;80:148- 158.

Reynolds, P.N., ei al. Nature biotechnology 200! ; l (9>:838-842.

Reynolds FN. et al. Mol Ther. 2000;2:562-578.

Rlvim JS. Methods Mol Biol 2013,946 383-393.

Rieger MA, et al. Cold Spring Harb Perspect Biol 2012;4,

Rivory LP, et al. Biochem Pharmacol 1996;52: 1103-1 1 1 1 ,

Roth, J.C., et ai Gem therapy 2008; i5(10):71 6-729.

Savoataos Ml. et al Geae TJher. 2002:9:972-9.

Schulze J, et ai. Bone 2010;46:524-533.

SohuSze J, et al. Cancer Lett 2012;317: 106-11.3.

Schwetzer L, et al BMC Cell Biol 20G8;9:4.

Seandel M, et al. Proc Natl Acad Sci U S A 2008: 105: 1 288- 19293.

Sentga B, et at. j Clin Oncol 201 1 ;29:3686-3694.

Seth P, et al. Biochem Biophys Res Commun. 2005:332:533- 1.

Shao L, et al. Cell Res 2009;19:296-306.

Sheldon H, et al. FASEB I. 2009;23:513-22.

Shindoh H, et al. J Toxicol Sci 20.1 1 ;36:4] 5 -422..

Shinkai Y_:, et al. Cell. 1992;68:855-67.

Sh.mo.zaki et al. Gene Ther. 2006; 13 52-59.

Shiozawa Y. et a!. i Clin invest 20! 1 ; ί 2 ! ; 1298- 1312.

Short JJ, et al. Mol Cancer Ther 2010;9:2536-2544.

Stnith-Berdaa S, et al. Cell Stem Cell. 201 1 ;8;72-83.

Song W, et al. Cancer Gene Ther, 2008: 55:667-75.

Sottnik JL, ei al. Cancer Microeirvimn 201 1 :4:283-297.

Stadtfe!d M, et al. Science 2008:322:945-949.

Su X, et al. J CIrn Invest 2012:122:3579-3592.

Sun Y^" et al. J Bone Miner Res 200 ;20:318-329.

Szymezak AL, et al. Na ioteehnol 2004;22:589-594.

Szymczak-Woikman AL, et al. Cold Spring Harb Protoc 2012;2012: 199-204.

Takayama K, et al Cancer Gene Ther 2007; 1 :105- 1 .16. Takebe et al. Nat Rev Clia Oncol 2011;8;97~106.

Tallone T. ei al. Proc Natl Acad Sci U S A.2001;98:7910-7915.

Tang, I., et al. Tramtatk ai research : (he journal of laboratory and clinical

2913;1ό1ί4};313~320.

Topf R et al Gene Ther 1998;5:507-513.

Triozzi PL, et al Expert Opin Biol Ther 2011 ;11 1669-1 76.

Valdex J , et al. Ceil Stem Cell 2012; i 1 :676-688.

van Beilo, J.H., et al. The Journal ofclinkal investigation 20l3;123(!);37-45. van Rooijeo, N., et al. Method* in molecular biology 2010;605:189-203.

Varda-Bloom N, et al. Gene Ther.2001 ;8:81 -27.

Voog J, et al. Cell Stem Cell 2010;6:103-115.

Waddrngton S , eial. Cell 2008:132:397-409.

WartgZ, et al, J Cell Biochem 2010:109:726-736.

Westermk WM, et at utat Res 2010;696:21-40.

White, KM., et ai. Journal of cardiothoracic surgery 20i3;8{! ):183.

Wilson A, et al. Cell 2008;.! 35: 1118-1129.

Wolff, G., et n\. Journal if Virology l97;71{l):624-629.

Xiong W, et al. J Viral 2006;80:27-37.

Xu, 2., et ai. Nature medki 201 ; l (4):452-457.

Yang C, et al Caacer Discov 2013;3:212-223.

Yang WY, et al World J Gastroenterol 2006;12:5331-5.

Ye H, et al Cancer Invest 20! 2:30:513-518.

Ye Q-F\ e al Asian Pacific Journal of Cancer Prevention 2012; 13:2485-2489. Zaiss, A. ., et al Journal of cellular biochemistry 2009;108(4}:778-790. Zeng L et al. Proc Natl. Acad Sci 11 S A 2009; 106:8326-8331.

Zeng Q, et al Ini J Nanoraedieme 2012;7:985-997.

Zhang X, ei al. PLoS Pathog.2012;8:el 002461.

Zhu TS, et al Cancer Res.2011:71 :60 1 -6072.

Claims

1. An adenovirus vector comprising a ROB04 enhancer/promoter operatively linked to a tratisgerte.

2. An adenovirus vector in accordance with claim I . wherein the traasgerte encodes a prodrug converting enzyme.

3. An adenovirus vector in accordance with claim 2, wherein the prodrug converting enzyme is a eytosine deaminase.

4. An adenovirus vector in accordance with claim L wherein the transgerte encodes a decoy receptor,

5. An adenovirus vector in accordance with claim 4, wherein the decoy receptor binds at least one angiocrine factor.

6. A adenovirus vector in accordance with claim I , wherein the transgene encodes a truncated CXCR4 receptor.

7. An adenovirus vector in accordance with claim i, wherein the ROB04 enhancer/promoter comprises a tissue-specific expression control element.

8. An adenovirus vector in accordance with claim i, wherein the R.OB04 enhancer/promoter comprises a ^"l^"et response element.

9. An adenovims vector in accordance with claim 1„ herein the ROB04 enhancer/promoter comprises a hypoxia-response element.

10. An adenovirus vector in accordance with claim I , wherein the ROB04

enhancer/promoter comprises a GABP-bmding element.

1 1. An adenovirus vector comprising:

a chimeric AD5-T4 phage fibrith shaft;

trimerteation domain displaying a myeloid cell-binding peptide ( BP); and a R.OB04 enhancer/promoter operativeiy linked to a transgene.

12. An adenovirus vector in accordance with claim I L wherein the transgeae encodes a prodrug converting enzyme.

13. An adenovirus vector in accordance with claim 12, wherein the prodrug converting enzyme is a eytosine deaminase,

1 . An adenovirus vector in accordance with claim 11 , wherein the transgene encodes a decoy receptor,

15. An adenovirus vector in accordance with claim 14. wherein the decoy receptor binds at least one angioerine factor.

16. An adenovirus vector in accordance with claim 11., wherein, the transgene encodes a truncated CXC 4 receptor.

17. A method of expressing a iransgene in an endothelial cell in vivo, the method, comprising administering to a mammal an adenovirus comprising a ROB04 enhancer/promoter operatively linked to a transgene.

18. A method of expressing a transgene in an endothelial cell in accordance with claim 17, wherein the transgene encodes a prodrug converting enzyme.

1 . An adenovirus vector in accordance with claim 18, wherein the prodrug converting enzyme is a cytosme deaminase.

20. An adenovirus vector in accordance with claim 17, wherein the transgene encodes a decoy receptor.

21. An adenovirus vector in accordance with claim.20, wherein the decoy receptor binds at least one angiocrine feeler.

22. An adenovirus vector in accordance with claim 17, wherein the transgene encodes a truncated CXCR4 receptor.

23. A method of mobilizing at least one of granulocytes, monocytes and lymphocytes from bone marrow, comprising administering to a mamma! an adenovirus comprisi g a R.OB04 enhancer/promoter operationally linked to a transgene encoding a truncated. CXCR4 receptor.

24. A. method of mobilizing cancer ceils in vivo, comprising administering to a mammal an adenovirus comprising a. ROB04 enhancer/promoter operationally linked to a transgene encoding a truncated C.XCR4 receptor.

25. A method in accordance with claim 24, wherein the cancer cells are comprised by bone marrow,

26. A method of selectively targeting endothelial eel Is, comprising administering to a mamma), an adenovirus comprising a chimeric AD5-T4 phage fibriiin shaft and trlmerizaiion domain displaying a myeloid cell-binding _.peptide ( BP), and an exogenous promoter operative ly linked to a transgene.

27. A method of selectively targeting endothelial cells in accordance with claim 26, wherein the promoter is a ROB04 enhancer/promoter.

28. A method of selectively targeting endothelial cells in accordance with claim 26, wherein the promoter comprises a Tet-responsive element.

29. A method of selectively targeting endothelial cells in accordance with claim 26, wherein the promoter comprises a hypos ia-responsive element.

30. A method in accordance with claim 26, wherein the endothelial cells are selected from the group consisting of brain ECs, kidney ECs and muscle EC's.

31. A method in accordance with claim 26, wherein the transgene encodes a truncated

CXCR4 receptor,

32. A method of treating a cancer, comprising:

administering to a mammal an adenovirus comprising a chimeric AD5-T4 phage ftbritin shaft and tri erization domain displaying a myeloid cell-binding peptide (MBP) and a nucleic acid sequence encoding a truncated CXCR4 receptor; and

administering a chemotherapeutic agent,

33. A method in accordance with claim 32, wherein the administering a chemotherapeutic agent consists of administering a therapeutically effective amount of the chemotherapeutic agent.

34. A method of treating a disease or disorder that activates angiogenesis in villous, endothelium, comprising:

administering to a mammal an adenovirus vector comprising a R.OB04

enhancer/promoter operatively linked to a transgene.

35. A method in accordance wit claim 34, wherein the disease or disorder that activates angiogenesis in villous endothelium is selected from the group consisting of inflammatory bowel disease regional enteritis, inflammatory bowel disease of the colon, infection with, toxi prodacing bacteria, and colon cancer precursor legions of multiple poly posis .

36. A method in accordance with claim 34, wherein the transgene encodes a secreted antiinflammatory cytokine decoy.

37. A method in accordance with claim 36, wherein the decoy is selected from the group consisting of soluble T F-alpha receptor, single chain anti-it I, single chain anti-lL I? antibody, a bacteria! anti-toxin, and an NAi .molecule targeting gene product induced by the act i vation of the WNT pathway in multiple polyposis.

38. A method in accordance with claim 35, wherein the toxin producing bacteria is selected form the group consisting of ^'Clostridium difficile, Cimiridmm bot iin n_^ Shigella,

39. A method of treating an inflammatory CNS disease in. a mammal, comprising:

administering to the mammal an Ad.MBP.CMV vector encoding a cytokine decoy .

40. A method in accordance with claim 3-9, wherein the inflammatory disease is selected from the group consisting of amyotrophic lateral sclerosis and multiple sclerosis.

41. A method of treating a degenerative disease in a mammal, comprising:

administering to the mammal an Ad.MBP.CMV vector -encoding a cytokine decoy.

42. A method in accordance with claim 41 _> wherein the degenerative disease is selected from the grou consisting of Alzheimer's disease and Parkinson's disease.

43. A method of stimulating appetite in a m mmal, comprising:

administering to the mammal an Ad.MBP.CMV vector encoding a secreted molecule thai affects the hypothalamic appetite nuclei

44. A method of inducing satiety in a mammal, comprising:

administering to the mammal an Ad.MBP.CMV vector encoding a secreted molecule that affects the hypothalamic appetite nuclei,

45. A method of treating myeiodysplastie syndrome in a mammal, comprising:

administering to the mammal an Ad.RGD.li5/iri3.ROB04 vector,

wherein the Ad/R.Gi.>.M5/H3.ROB04 vector produces at least one antiinflammatory molecule.

46. A method of treating a genetic disease selected from the group consisting of hemophilia and. sickle cell anemia in a mammal, comprising:

administering to the mammal an Ad.RGD.H5/H3.ROB04 vector,

wherein the A&RGD.H5/H3. OBD4 vector produces at least one antiinflammatory molecule.

47. A. method of treating a cancer in a mammal, comprising:

administering to tire -mammal an Ad.RGD.R5/B3.ROB04 vector,

wherein the Ad.RGD.H5/H3.ROB04 vector produces at least one molecule selected from the group consisting of a molecule that mobilizes metastatic cancer or leukemic stem cells and a molecule producing a chernoiherapeutic prodrug converting enzyme.